INTRODUCTION:

The development and spread of drug resistance among bacteria have increased at an alarming rate over the past several decades¹. These days, multidrug-resistant microbes are spreading and pose a major risk to human health and restrict the effectiveness of antimicrobials in the treatment of infectious diseases^2,3.

Enterococcus faecalis a gram-positive opportunistic pathogen that is part of microbiota in animals and humans but can also be found in environments like water and soil^4,5,6. Enterococcus faecalis is an important human pathogen and is responsible for 85–90% of all enterococcal infections such as urinary tract infection, bacteremia, endodontic infections, wounds, and endocarditis^7,8,9. Multidrug-resistant enterococci have become a major problem in recent times and have been reported increasingly around the world^10,11,12,13. Virulent bacteriophages (phages) are obligate intracellular viruses that infect and reproduce only inside bacterial cells without invading other cells and have been used ever since for therapeutic purposes to the treatment of bacterial infections and destruction of multidrug-resistant (MDR) bacteria^14,15. 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 future¹⁶. Phages are used as therapeutic drugs in the management of chronic and acute infections in urology, stomatology, dermatology, surgery, and pediatrics¹⁷. Resistance to phages is a challenging problem which needs addressing. Bacteria have developed several different mechanisms for self-defense against bacteriophages, including phage adsorption prevention, restriction enzymes, DNA injection, and CRISPR/Cas systems^18,19,20.

A main benefit of phage therapy over traditional therapies is the applying of phage cocktails to avoid the creation of resistance²¹. The use of many phages, that each target with a specific receptor and also with a particular genetics clade would increase the efficiency to minimize loss with adsorption or mechanisms for host genetic defense²². Using phages to tweak the microbiome to better resemble that of a healthier individual, enhancing disease outcomes, and recent work into fecal transplants has pointed to the possible involvement of phages in the efficacy of such treatments ²³ Phages are assumed to be harmless, however, and billions passed via the human body daily²⁴.

Phage endolysins or lysins are extremely effective particles that are being optimized for this very purpose for millions of years²⁵. Endolysin are enzymes that destroy bacterial peptidoglycan after the phage reproduction is complete²⁶. The main advantages of endolysin: high specificity for their target bacteria, non-toxicity to eukaryotic cells and very low risk of developing resistance due to their highly conserved target bonds with peptidoglycan (PG)²⁷.

Accordingly, this study was conducted to isolate, purified, and optimized lytic phages specific to Enterococcus faecalis. Subsequently, a phage cocktail was formed and a phage-endolysin was extracted in which its antibacterial activity was tested.

MATERIALS AND METHODS:

Bacterial Collection, Identification and Antibioresistant Pattern:

Thirty E. faecalis isolates were collected (EF1-EF30) from The Teaching Laboratories in the Medical City, Laboratories of Al-Kindy Teaching Hospital and Hilla Teaching Hospital, in the period from Dec. 2019 to Feb. 2020. The specimens were from patients with different infectious diseases such as urinary tract infection (UTI), diabetic foot, septicemia, and wound infections. A re-identification was carried out by the VITEK 2 Compact system.

All isolates were examined by VITEK 2 System for species-level identification and to evaluate their antibiotics sensitivity pattern. The bacterial samples were prepared following the manufacturer’s instructions. Overnight bacterial cultures in Blood agar (Himedia, India) were used. The VITEK 2 Compact system uses a Gram-positive cocci identification kit (ID-GP) as a fluorogenic recognition method in less than eight hrs and a turbidimetric susceptibility testing in less than 18 hrs for bacterial sensitivity.

Isolation and Optimization of the Specific Bacteriophage:

Ten phage crude suspensions were collected from the ecosystem in the city of Baghdad such as: chicken litters, sewage, cattle farms sewage, wastewater, farm soil, feces of sheep, and goat feces during one week in January 2020. Overnight bacterial broth (100 µl) was mixed with 3 ml of crude samples and incubated at 37C˚ overnight until obtaining the specific lytic phage by the top layer assay²⁸. The clearest and largest plaque had been selected. The tiny and turbid plaques had been optimized by attempting to perform serial passage in the top layer plaque assay method according to²⁹.

The Biokinetics of the Isolated and Optimized Bacteriophages:

The biokinetics of the optimized lytic phages were measured according to²⁹. The plaques were examined to calculate the burst time, the burst size, and the infective percentage as follows:

Burst Time (BT): the time required to burst and leave the bacterial cells by the infecting phages. So is a period for certain dilution before a rapid rise in the number of progeny.

Burst size (BS): number of new phage progenies per one target bacteria cell. The burst size determined by dividing the plaques numbered in the post burst time over the plaques numbered of in pre burst time.

The infective percentage (IP %): is a percentage for the lytic phages which invading the bacterium. The IP % is measured by dividing the pre-burst time plaques number (from top layer assay) over the number of bacteria that used during this method.

The Coverage and the Resistance Rate of the Formed phage cocktail against E. faecalis:

The phage cocktail was formed by the addition of 100µl of 10⁹ PFU/ml of each phage stock in 15ml tube. In order to determine the coverage rate of the phage cocktail, 10 new samples of E.faecalis randomly from patients in the Medical City Hospital. The specimens collected were not biased against a particular illness. Ten µl of the phage cocktail suspension were spotted on the bacterial lawn using the top layer assay. After dryness, all plates were incubated at 37C˚for overnight. The next day, if the lysis zones had appeared meant that the bacterial isolates were susceptible to the phage cocktail.

The coverage rate was calculated according to the following formula:

Number of bacteria lysed by cocktail

Coverage rate = ------------------------------------------------------- × 100%

Total number of bacteria

The bacterial resistance rate was calculated as the following formula:

Number of resistant colony per phage lytic spot

The Resistance Rate = ---------------------------------------------------------

Number of colony formed from the same size cut

of the lawn

Endolysin Extraction:

The lytic enzyme from the specific phage was extracted according to²⁹ with some modification. E.faecalis (EF13) in 100ml of Nutrient broth medium (Himedia, India) were cultured and incubated overnight at 37C˚. The next day, 250ml of sterile Nutrient broth medium were added to the bacterial growth and incubated for another three hours. Ten ml of the specific phage at titer 1×10⁹ PFU/ml were mixed with the bacterium then put directly in ice for 20 min. The mixture was centrifuged at 10,000 g for 20 min and the sediment was collected. The sediment was suspended in 10ml of 0.05 M phosphate buffer saline (Chemical, point Germany) + 5mg deoxyribonuclease (REF, Germany) and incubated for 60 min at 37C˚. EDTA (RPI, USA) at (0.005 M) was added and centrifuged at 10,000g for 60 min then the supernatant was taken. Ammonium sulfate (Merck, Germany) was added to 85% saturation and incubated for overnight at 4C˚. The next day, the suspension was centrifuged at 10,000g for 60 min and was re-suspended in five milliliters of 0.05 M phosphate-buffered saline at pH 7.5. Dialysis against 200ml of 0.05 M phosphate buffer saline was done. The buffer was changed every two hrs for two times and kept overnight at 4C˚. The dialysis solution was added to Sephadex G.100 column chromatography (GE healthcare, USA) (18 × 0.5cm) in 20ml of 0.1M phosphate-buffered saline at pH 7.5 as eluent. One ml of elute was collected in 12 Eppendorf tubes every five min. From each Eppendorf tube, 10µl of the elute were dropped onto E.faecalis lawns to catch the tube with the endolysin lytic activity.

The endolysin antibacterial activity against E.faecalis was measured by turbidity reduction assay: the overnight bacterial cells were centrifuged at 4000g for 30 min and resuspended with phosphate-buffered saline pH (7.5). At room temperature, 30µl of elute-containing endolysin was added to 270µl of the bacterial broth. Then at 600 nm, the optical density was measured by spectrophotometer every 10 min for one hr. Later, by using the control standard curve, the absolute delta for the following OD/min, Bacterial log reduction, and Bacterial concentration (CFU/ml) were measured according to the below formulae:

ΔOD = OD (on time zero) - OD (on time 60)

Δ Bacterial log reduction = Bacterial log (on time zero) – Bacterial log (on time 60)

Δ Bacterial concentration (CFU/ml) = CFU/ml (on time zero) – CFU/ml (on time 60)

The control standard curve was plotted for E.faecalis in Nutrient broth. Serial dilutions for the overnight cultured bacterium were done (10^-1 to 10^-6). The absorbance values were measured at 600nm for all the dilutions and the standard curve was plotted.

The Antibacterial Spectrum of Endolysin:

The activity of the extracted endolysin was tested against 20 E.faecalis isolates. Ten micro liter of the endolysin was spotted on overnight bacterial lawns. The formed lytic zone after overnight incubation was a marker for endolysin activity. The coverage rate was calculated according to the previously mentioned formula.

Endolysin Molecular Weight:

In order to determine the molecular weight of the extracted endolysin a SDS-PAGE method was used as follows:

The protein sample (endolysin) was denatured for five min at 95C˚ by mixing 50µl of the sample protein with 50µl of the loading buffer (SDS + β-mercaptoethanol (Bio-Rad, Japan) + bromophenol blue (Gunduzkimya, Turkey) + glycerol (BHD, England). The electrophoresis system was adjusted and washed by 1 X Tris-glycine SDS buffer (RPI, USA). Twenty microliters of the protein sample and the marker were loaded into the wells in a consecutive order. The electrophoresis was run in 30% acrylamide gel at 100 volt for one hr till the bromophenol blue dye reached the bottom of the gel. The gel was removed from the electrophoresis chamber and transferred to another chamber containing Coomassie blue (Bio-Rad, Japan) staining solution. The gel was incubated with a staining solution for 30 min at 50C˚. Later, the gel was transferred to a re-staining solution which changed every 15 min until the bands were visualized. The concentration of the extracted endolysin was recovered by the Biuret method.

RESULTS:

E. faecalis Isolates and the Antibioresistant Pattern:

Thirty isolates of E. faecalis studied in this research. E.faecalis isolated from urine showed the highest percentage (36.66%) followed by those isolated from blood (23.33%) and wound swab (13.33%). While the isolates from diabitic foot infection revealed (10%), CSF (6.66%), vaginal swab (6.66%), and sputum (3.33%). Thus, the most frequent isolates of E.faecalis were from UTI then septicemia and wound infection. The macroscopic identification on Blood agar disclosed pale, milky, and non-hemolytic colonies, the microscopic examination uncover G+ve cocci in short chains, clusters, single cocci, and diplococci.

The VITEK system results revealed that all isolates (EF1 to EF30) showed 100% resistance to Cefoxitin, Erythromycin, Amikacin, and Ceftriaxone, while they showed 100% sensitivity to Tigecycline.

Characteristics of the Isolated and Optimized Phages:

The plaque assay of the isolated phages showed that plaques clarity ranged from clear, semi-clear, turbid, to semi-turbid. The size of the plaques was from 0.2mm to 1mm. The margin cut was different and ranged from regular to irregular, and plaques shape from oval to circular, Table (1). The titers of the isolated phages were varied from 10⁴-10⁹PFU/ml. About 75 phages specific for E.faecalis were isolated. Most of the isolated phages were highly lytic and produced obvious inhibition zone on target E.faecalis, Figure (1).

Figure (1) E.faecalis specific phages which isolated from the environment A) purified by 0.2µm filter syringe and chloroform. B) Purified by chloroform.

The Biokinetic characteristics of the Isolated and Optimized Phages:

In this study, five specific phages for different E.faecalis isolates were randomly selected to give representative values of the biokinetic characteristics. The results showed that the average of the burst time was 55 min ranging from 45 to 65 min; furthermore, the average of the burst size was 135 progeny, the minimum burst size was 120 and, the maximum burst size was 150 progeny. Thus, the average of the infective percentage was 86% ranging between 78% and 94%, Figure (2).

Figure (2) The burst time (BT), burst size (BS), and the infective percentage (IP %) of the randomly selected optimized phages.

The Specificity of the Formed Phage Cocktail to E.faecalis and its Coverage Rate:

All the isolated phages formed visible inhibition zones in the early stage when tested on the bacterial lawns. The phage cocktail was formed by mixing phages specific for E.faecalis with titer 10⁶PFU/ml. The results showed that all the bacteria except EF5, EF6, and EF8 were specific to over than one phages.

Additionally, all the isolated bacteria were 100% sensitive to the lytic phages applied with zero resistant bacteria colonies.

The formed phage cocktail was capable to create inhibition zones on the most bacterial isolates' lawns. The phage cocktail was shown to be able to lyse six out of ten E.faecalis and thus the coverage rate was 60%.

Determination of the Phage Endolysin Activity against E.faecalis:

Endolysin was successfully extracted by using Sephadex G100 column chromatography from E.faecalis bacteriophage (EF13P3). The elute was collected in (12) tubes, only two tubes (7&8) gave positive results on the corresponding bacterial lawns, i.e. (EF13), Figure (3).

The antibacterial activity was evaluated by the turbidity reduction assay depending on the O.D level which measured every 10 min. A clear decline in the optical density was manifested that ranged from (0.597 to 0.029), the control standard curve was plotted to EF13 serial dilutions in nutrient broth. To signify the differences in the level of the bacterial growth after treatment with endolysin, the absolute delta was measured. Treating E.faecalis 13 (EF13) by endolysin revealed a decline in the bacterial turbidity when measured by spectrophotomer for one hr. Significant differences were found between the OD of the bacterial suspensions at time zero (0.597) and that at time 60 min (0.029). Similarly, the differences between the bacterial log at time zero (6.75) and at time 60 min (3.11) and the bacterial concentrations at time zero (5x10⁷ CFU/ml) and at time 60 min (1x10⁴ CFU/ml) were clear. Thus, the bacterial growth was reduced by (3.63 log) and (4x10³ CFU/ml), Table (2).

Figure (3) The activity of the extracted endolysin against the phage specific E.faecalis (EF13). A) endolysin in tube number (7). B) endolysin in tube number (8).

The Antibacterial Spectrum of the Extracted Endolysin:

The coverage rate of the extracted endolysin was measured against 20 E.feacalis isolates (EF1 to EF20). From the twenty tested bacteria 18 were completely sensitive to the extracted endolysin to give a coverage rate of (90%). The bacterial isolates were as follows: 6/20 (30%) urine specimens, 5/20 (25%) blood, 3/20 (15%) wound infection, 2/20 (10%) from diabetic foot ulcer, and one isolate (5%) from each of sputum specimen and vaginal swab. All the tested bacteria were completely sensitive to the extracted endolysin except two

The Characteristics of the Extracted Endolysin:

The SDS-PAGE electrophoresis revealed clear bands of the extracted endolysin from tubes number (7) and (8). The bands molecular weight found to be as 48 kDa, Figure (4).

Figure (4) shows the molecular weight of extracted endolysin in SDS-PAGE electrophoresis in which (lane M) is the protein molecular marker, (lane 1) endolysin from tube number seven, (lane 2) endolysin from tube number eight.

DISCUSSION:

With the emergence of antibiotic-resistant (especially vancomycin-resistant) E. faecalis, alternative intervention tools or strategies have been pursued for treating these drug-resistant Enterococci infection in future³⁰. Under these circumstances, a therapy based on phage or its lytic component acquired reconsideration³¹.

In the current study the majority of E.faecalis isolates were from urine (36.6%) to be followed by those isolated from blood (23.33%) and wound infections (13.33%). These findings were similar to other studies conducted in Iraq and Saudi Arabia which found that the most predominant E.faecalis isolates were from urine^32,33. Additionally, many studies conducted in India and USA found that E.faecalis was isolated more frequently from urinary tract infections, blood and wound infections ^34,35,36.

The isolated E.faecalis was 100% resistant to Cefoxitin, Erythromycin, Amikacin, and Ceftriaxone and 100% sensitive to Tigecycline. This antibioresistant profile was similar to a recent study in Iraq by Al-naqshbandi et al. in 2020 which explored the fact that E.faecalis isolates were (100%) sensitive to Tigecycline and the resistance were as (100%) to Ceftriaxone and Cefotaxime, 90% Erythromycin and 80% Tetracycline³⁷. Exploring the antibioresistant pattern can give a great assistant to the physicians trying to treat this commonly attributed bacterium in Iraq. All the isolated E.faecalis was reported as MDR in this study. These findings were in harmony with other similar studies in Iraq³⁸ and Egypt ³⁹. In contrast, a study in Italy by Bertelloni et al, showed that (53%) isolates of E.faecalis were classified as multidrug-resistant (MDR) and (5.2%) strains as possibly extensively drug-resistant (XDR)⁴⁰. These findings may highlight the potential of this bacterium to be converted from MDR to XDR in the future in Iraq and other parts of the world.

Specific E.faecalis phages were isolated from environmental sources in the current study. Like other previous reports about E.faecalis phage sources⁴¹, the main two sources for the isolated phages were sewage and waste water. The biokinetic assay for five selected optimized phages revealed that the burst time average was (55 min). Therefore, 55 min was the time required to rupture the bacteria by the lytic phages. The average of the new progeny (burst size) of the lytic phages was (135) in each rupture or burst time. From this new progeny about 86% (infective percentage) were able to invade the new specific bacteria. As no previous study was carried to record the biokinetic characteristics of E.faecalis isolated phages, a comparison with other phage characteristics were tried to give an idea about the current isolated phage potency. A study in Iraq on Acinetobacter baumannii by²⁹ found that the burst time average for the specific isolated phages was 73.5 min and the average of the new progeny (burst size) was 245 progeny with 85.45% infective percentage.

The resistance challenge experiments of this study showed that the rate of the resistance of the E.faecalis bacteria was lower and were more successful than the single phage therapy. In this study a phage cocktail was formed and tested against E.faecalis. Twelve phages were tested against 10 E.faecalis isolates. No sign of resistance was observed in the developed lytic zone by the formed phage cocktail. The absence of resistant colonies in the lytic zone came to the fact that the ten MDR isolates were completely sensitive to the phage cocktail. In contrast to other study in Iraq, which tested the activity of its formed phage cocktail against Acinetobacter baumannii. This study recorded some level of resistance among the tested bacteria (5 out of 23 isolates, 21.7%). The phage cocktail was tested against these new 10 MDR isolates in order to recover the coverage of its activity against different strains of E.faecalis. A coverage rate of 60% was recorded of the formed phage cocktail against E.faecalis. These results found to be partially similar to another study that tested the coverage rate of its formed phage cocktail against Acinetobacter baumannii which was 70%²⁹. The coverage rate results encourage the usage of phage cocktail in treating MDR bacteria over the specific phage alone.

Endolysin was successfully extracted by using a gel filtration column chromatography from E.faecalis bacteriophage. The antimicrobial activity of the purified protein was demonstrated by the turbidity reduction assay depending on the O.D level which measured every 10 min (ΔOD = 0.568) and found to reduce the bacterial growth by (3.63 log). The finding of this study was in compact with other study which found that E.faecalis phage endolysin has a molecular mass of (46 kDa)⁴². While other study in USA recorded a molecular mass of 27 kDa for E.faecalis endolysin⁴³. Twenty bacterial isolates of MDR E.faecalis were tested to disclose the antibacterial spectrum of the extracted endolysin and tested the coverage rate of this lytic enzyme. The extracted endolysin found to be effective against 90% of the tested MDR E.faecalis isolates. The finding was in agreement with a study that showed the bactericidal of endolysin against E.faecalis was able to kill 32/36 of a panel of diverse E.faecalis isolates⁴⁴. This wide-range of activity overcome the coverage rate of the phage cocktail (60%) and increase the hope to use this effective antibacterial therapy to treat MDR E.faecalis infections.

Further analysis is crucially acquired for the extracted endolysin. The analysis must include the antibacterial activity on other Gram-negative genera, in vivo antimicrobial efficacy of the phage lytic enzyme on laboratory animals. In addition, the genetic profiles to be analyzed in order to give complete overview on this promising antibacterial agent and open the gate for future clinical trial.

CONCLUSION:

The study concluded that all the isolated E.faecalis were MDR which represented different serious clinical conditions like UTI, septicemia and wound infections. All the isolates found to be completely sensitive to (Tigecycline) and resistant to (Cefoxitin, Erythromycin, Amikacin, and Ceftriaxone). The specific phages against the isolated E.faecalis were mainly from sewages and waste water which reflected the natural inhabitant of this bacterium; i.e., gastrointestinal tract of human and animal. As far as we know, no previous study in Iraq has isolated the specific phages against local isolates of E.faecalis and its effective endolysin. The phage cocktail gave distinct zone of inhibition with no resistant colonies against them. The phage cocktail could cover 60% of E.faecalis bacterium. The proteolytic enzyme of the specific phage; i.e., endolysin was extracted by the gel filtration method and demonstrated a well-defined zone of inhibition when tested on its specific E.faecalis. Interestingly, the extracted endolysin found to be effective against 90% of E.faecalis isolates which reflected its broad spectrum activity. These results may open the gate for successful and inexpensive method of therapy to treat MDR E.faecalis and overcome the rapid development excessively resistant strains.

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Received on 03.10.2020 Modified on 28.10.2020

Research J. Pharm. and Tech. 2021; 14(9):4547-4554.

DOI: 10.52711/0974-360X.2021.00791

Phage symbol	Plaque size(mm)	Plaque clarity	Plaque shape	Margin cut
EF2P2	0.2	Semi-clear	Round	Irregular
EF2P4	0.3	Clear	Oval	Irregular
EF4P9	0.3	Semi-clear	Oval	Irregular
EF3P6	0.4	Semi-turbid	Oval	Regular
EF6P12	0.4	Semi-turbid	Round	Irregular
EF4P8	0.5	Clear	Round	Regular
EF1P1	0.5	Turbid	Round	Regular
EF4P10	0.6	Semi-turbid	Oval	Regular
EF4P7	0.7	Turbid	Round	Regular
EF3P5	0.8	Semi-turbid	Round	Regular
EF13P3	1	Semi-clear	Round	Irregular
EF5P11	1	Clear	Round	Regular

Control EF13		Endolysin EF13 treated bacteria
Concentration (CFU/ml)	O.D	Time (min)	O.D	Concentration (CFU/ml)	Log
10⁸x1	0.82	0	0.597	5x10⁷	6.7505731
10⁷x1	0.63	10	0.482	1x10⁷	6.0143086
10⁶x1	0.49	20	0.361	1x10⁶	5.2396303
10⁵x1	0.25	30	0.244	3x10⁵	4.4905612
10⁴x1	0.06	40	0.187	1x10⁵	4.1256301
10³x1	0.01	50	0.095	3x10⁴	3.5366185
10²x1	0.005	60	0.029	1x10⁴	3.1140667
			ΔOD = 0.568	ΔCFU/ml =4x10³	Δlog =3.6365064