Isolation and Characterization of Bacteriophages from Wastewater Sources on Enterococcus spp. Isolated from Clinical Samples | SciTechnol

Journal of Virology & Antiviral Research.ISSN: 2324-8955

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Research Article,  J Virol Antivir Res Vol: 10 Issue: 2

Isolation and Characterization of Bacteriophages from Wastewater Sources on Enterococcus spp. Isolated from Clinical Samples

Yara Elahi1, Jamileh Norouzi2 and Ramin Mazaheri Nezhad Fard3*

1Department of Genetics, Faculty of Life Sciences, Tehran North Branch Islamic Azad University, Tehran, Iran

2Department of Microbiology, Faculty of Life Sciences, Tehran North Branch Islamic Azad University, Tehran, Iran

3Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

Corresponding Author:
Ramin Mazaheri Nezhad Fard
Department of Pathobiology
School of Public Health
Tehran University of Medical Sciences
Keshavarz Blvd., Pour Sina St.
Tehran, Iran, PO Box: 1417613151

Received: December 22, 2020 Accepted: March 23, 2021 Published: March 30, 2021

Citation: Elahi Y, Norouzi J, Fard RMN. (2021) Isolation and Characterization of Bacteriophages from Wastewater Sources on Enterococcus spp. Isolated from Clinical Samples. J Virol Antivir Res 10:2.


Due to dramatic increases in bacterial antibiotic resistance in recent decades, use of antibiotics to treat infections is not thoroughly effective. A group of these bacteria, enterococci, are highly resistant to common antibiotics, especially vancomycin. Therefore, researchers have used bacteriophages, as drug alternatives, to treat bacterial infections resistant to multiple antimicrobials. The most important reasons for using bacteriophages to treat antimicrobial-resistant strains include relative safety of the bacteriophages compared to chemical antimicrobials, zero or low resistance of the bacterial strains to their host-specific bacteriophages and ineffectiveness of the bacteriophages on eukaryotic cells. Thus, the major aims of this study were to isolate and identify bacteriophages of municipal wastewaters on antibiotic-resistant clinical enterococci. After isolation of the bacteriophages, their efficiency on various enterococcal species was investigated. In general, three bacteriophages were isolated, including those of Myoviridae, Siphoviridae and Inoviridae families, which were isolated on an Enterococcus faecium strain. The bacterial strain was partially sequenced and further studied using transmission electron microscopy. In conclusion, bacteriophages can be isolated from biological sources. The bacterial viruses are suggested as viable alternatives to antimicrobials because of the current bacterial multiple resistance to these biochemical agents. Further studies are necessary to verify effects of bacteriophages on multiple resistant pathogens

Keywords: Enterococcus spp; Bacteriophages; Clinical samples; Wastewaters


Enterococcus spp; Bacteriophages; Clinical samples; Wastewaters


Enterococcus spp. are Gram-positive bacteria and gastrointestinal tract colonizers that opportunistically colonize wounds and bloodstream, causing life-threatening infections such as bacteremia and endocarditis [1,2]. These bacteria are particularly associated with central-line associated bloodstream infection (CLABSI), a type of hospital-acquired infection (HAI) that arises from use of central venous catheters. Enterococci are linked to 18% of CLABSIs in the United States [3]. The bacteria are part of the normal intestinal flora of mammals, birds and humans. Of all enterococcal species, Enterococcus faecalis and E. faecium are the most commonly identified species in human samples, whereas E. gallinarum and E. casseliflavus are less identified [4]. Enterococci are the cause of nosocomial infections, most frequently associated with intraabdominal, pelvic, catheter, surgical, central nervous system (CNS) and urinary tract (UT) infections and endocarditis [5]. Enterococci are shown to include relatively high antibiotic resistances. It has been reported that enterococci from livestock and companion animals can be transmitted to humans via direct contacts [6]. In terms of public and animal health, it is important to prevent transmission of multidrug-resistant (MDR) enterococcal strains between animals or from animals to humans [7]. A previous study reported that animals treated with antibiotics in intensive care unit (ICU) were sources or zoonotic transmitters of MDR Enterococcus species [8]. In USA, 35–75% of enterococcal infections are caused by E. faecium; from which, a majority are vancomycin-resistant [9]. In addition to threats posed by VRE, MDR enterococci serve as reservoirs for horizontal gene transfer (HGT) of antibiotic resistance to other pathogens; as verified by reports of vanA transfer from Enterococcus species to Staphylococcus aureus [10].

Bacteriophages (phages) are viruses capable of infecting and replicating within the bacterial cells. They are the most abundant and ubiquitous entities on Earth, playing important roles in microbial physiology, evolution, population dynamics and therapeutics. Bacteriophages replicate through two primary life cycles or dynamic mechanisms, which are important for their therapeutic uses. Virulent or obligate lytic phages infect and quickly kill their bacterial hosts, whereas temperate or lysogenic phages may either stably integrate into their host genome or enter a lytic life cycle [11]. Phage therapy is described as direct administrations of lytic phages to patients to lyse bacterial pathogens causing clinical infections [12]. Recently, phage therapy has been interested as an alternative antimicrobial strategy to treat antibiotic-resistant biofilm-forming pathogens. For example, topical phage therapy is now considered as a good option in infections of burn wounds primarily caused by Pseudomonas aeruginosa and Staphylococcus aureus [13]. Therefore, the major aims of this study were to isolate and identify phages from municipal wastewaters on antibiotic-resistant clinical Enterococcus strains.

Materials and Methods

Bacterial strains

Bacterial strains were isolated from clinical patients referred to Imam Khomeini Hospital, Tehran, Iran, 2019–2020. The current study was approved by the Ethical Committee of Tehran University of Medical Sciences, Tehran, Iran (approval no. IR.TUMS.SPH. REC.1397.139). Relative information of the patients were collected using questionnaires. Blood samples were immediately cultured on bile esculent agar and incubated at 37 °C for 24 h. Isolates were verified using morphological, biochemical and molecular techniques. First, isolates were Gram stained and studied using direct light microscope. Then, isolates were biochemically identified using arabinose fermentation test, salt tolerance test, optochin susceptibility test, CAMP test and PYR test. Molecular identification of the isolates was carried out through amplification of the tuf (elongation factor Tu) gene using polymerase chain reaction (PCR) technique. An amplicon of each isolate were sequenced using Sanger sequencing method.

Sanger sequencing of the bacterial genome

To sequence the bacterial genome, a colony of the bacteria was dissolved in sterile distilled water using sterile microtube. The microtube was incubated at 90 °C for 30 min to extract the genome. After centrifugation of the solution, concentration of the extracted DNA was measured and the 260/280 and 260/230 nm ratios were calculated using NanoDrop One (Thermo Fisher Scientific, USA). Furthermore, genome was evaluated using agarose gel electrophoresis. The bacterial genome was sent for Sanger sequencing and the tuf gene partial sequence result was annotated in DNA Data Bank of Japan (DDBJ) under the accession number of LC580430.

Bacterial antimicrobial susceptibility assessment

The antimicrobial susceptibility patterns of the isolates were assessed using Kirby-Bauer method and the following antimicrobials of ceftriaxone (30 μg), cefoxitin (30 μg), clindamycin (2 μg), erythromycin (15 μg), linezolid (30 μg) and vancomycin (30 μg).

Detection of bacteriophages

Briefly, a colony of the bacterial sample was inoculated into 5 ml of TSB liquid media and incubated at 37 ℃ overnight. Then, various wastewater samples were collected for phage isolation. Wastewater samples were centrifuged at 700 g for 10 min. After centrifugation, supernatant was filtered using 0.45-ml syringe filters. The bacterial samples in TSB were mixed with filtered sewage samples and 5 ml of BHI broth media and incubated at 37 °C overnight. A colony of the bacterial sample was inoculated into TSB media and incubated at 37 °C overnight. Incubated culture was centrifuged at 700 g for 10 min. Supernatant was filtered through 0.45-ml syringe filters; then, 300 μl of chloroform were added to the filtrate with agitation and set for 10–15 min. This was centrifuged at 1600 g for 5 min and the supernatant was filtered through 0.45 ml syringe filters. Then, 4.5 ml of 0.75% TSA, 360 μl of the filtered wastewater sample and 160 μl of overnight bacteria were poured into a sterile microtube and set for 10 min with agitation. This was mixed well with top agar and poured onto the plates. After 10 min of setting at room temperature, plates were incubated at 37 °C overnight. After overnight incubation, plates were studied for the presence of the phage plaques.

Plaque purification and propagation

To purify phages, a plaque was selected from the plate and transferred into a SM buffer containing microtube using Pasteur pipettes. The microtube was centrifuged to precipitate debris and the liquid was filtered using syringe filters. The liquid was added to the broth media and after 24 h, 360 μl of TSB media and 160 μl of the bacteria in logarithmic phase were added to 0.75% TSA top agar. After 24 h, the previous steps were repeated three to five times to purify phages. Plaques achieved from the final purification steps were stored at 4 °C after centrifugation and filtration in SM buffer.

Bacteriophage titration

To prepare phage samples for TEM imaging and whole genome sequencing, phages were first diluted. For the titration of phages, nine microtubes were prepared for 100 to 10-8 serial dilutions. First, 900 μl of SM buffer were added to each microtube. 100 μl from the phage samples were transferred into the microtube of 100 dilution. Then, 100 μl from this solution were transferred into the microtube of 10-1 dilution. This method was repeated to prepare all dilutions to 10-8. Then, 100 μl of the diluted solution and 200 μl of the overnight cultured bacteria were added to TSA top agar and poured onto the plates. After 24 h, number of plaques in each plate was counted and the phage titers were calculated using the following formula:


Where, C was the phage titer, n was the plaque count, d was dilution factor and v was the volume of dilution added.

Bacteriophage genome extraction

Briefly, 10 μl of the phage solution were filtered and mixed with DNase 1 and RNase A. This was incubated at 37 °C for 30 min and then mixed with 4 μl of 20% PEG 6000. The mixture was incubated on ice for 1 h and centrifuged for 30 min. The supernatant was discarded and the precipitate was dissolved in 600 μl of SM buffer. Then, 25 μl of phenol, 24 μl of chloroform and 1 μl of isoamyl alcohol were added to the solution. The solution was centrifuged for 20 min and then mixed with 600 μl of chloroform and re-centrifuged for 20 min. Then, 600 μl of isopropyl alcohol were added to the mixture and incubated at -80 °C for 12 min. After 20 min of centrifugation of the solution and discarding of the supernatant, 700 μl of ethyl alcohol were added to the precipitate. After 20 min of centrifugation of the solution and discarding of the supernatant, the precipitate was dried under the hood for 10 min and then dissolved in 50 μl of TE buffer.

Bacteriophage genome typing

First, purity of the extracted phage genome was measured at 260/280 nm and 260/230 nm. Then, 1 U of each DNase 1, RNase A and endonuclease S1 enzymes was added to 1 μg of the extracted genomes and incubated at 37 °C for 2–3 h. In the next step, 10 μl of each reaction were electrophoresed on 1% agarose gels and the resulting bands were analyzed under UV light.

Host specificity of bacteriophages

To investigate host specificity of the phages, the isolated phages were examined on Escherichia coli (ATCC 35218), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 29213), Salmonella enterica (ATCC 13076), salmonella typhimurium (ATCC 14028) and Streptococcus dysgalactiae (ATCC 27957). standard strains. These strains were kindly provided by the Faculty of Veterinary Medicine, University of Tehran, previously characterized and used in other studies.

Transmission electron microscopy

To prepare phage samples for the electron microscopy, phage plaques were diluted to 10-8 in sterile microtubes using SM buffer. Microtubes were centrifuged and the supernatants were transferred to fresh sterile microtubes. Then, 200 μl of each sample were used for phage staining. For the phage staining, 2% uranyl acetate dye and carbon-coated grids were used. After staining, samples were studied using transmission electron microscopy (TEM) (Philips EM208S, Netherland) at 100 KV.


In general, 25 Enterococcus strains were isolated from the clinical samples in this study. The enterococcal species, including E. fecalis, E. faecium and E. gallinarum, were isolated from blood samples. In this study, most of the isolated strains were resistant to vancomycin, erythromycin and clindamycin. Enterococcus faecium EntfacYE was sensitive to linezolid and resistant to vancomycin, erythromycin, clindamycin, ceftriaxone and cefoxitine.

The tuf gene partial sequencing results verified primary characterizations of the isolated bacteria. Overall, three phages were isolated on one of the enterococcal isolates using two various wastewater sources of public ponds in Tehran (Figure 1). Two phages included isometric shapes, one with a long flexible tail (Siphoviridae) (Entfac.YE1) and another one with a non-flexible tail (Myoviridae) (Entfac.YE2) (Figures 2 and 3). The third phage was filamentous (Inoviridae) (Entfac.YE3) (Figure 4). The two tailed phages contained double-strand DNA (dsDNA) and the filamentous phage singlestrand DNA (ssDNA) genomes. In host specificity assay, all the three phages were able to lysis Streptococcus sp. as well (Table 1).

Figure 1: Lysis of the Enterococcus spp. by the bacteriophages.

Figure 2: Entfac.YE1 enterococcal bacteriophage. An isometric head with flexible tail.

Figure 3: Entfac.YE2 enterococcal bacteriophage. An isometric head with non-flexible tail.

Figure 4: Entfac.YE3 enterococcal bacteriophage. A long filamentous bacteriophage.

Table 1. Characteristics of the isolated enterococci bacteriophages

Bacteriophage Nomenclature Head length (nm) Head width (nm) Tail length (nm) Tail width (nm) Genome
Entfac.YE1 Siphoviridae 96 90 192 13 dsDNA
Entfac.YE2 Myoviridae 81 81 121 13 dsDNA
Entfac.YE3 Inoviridae N/A N/A 800 15 ssDNA


In this study, bacterial strains were isolated from human blood clinical samples. Of 25 isolated enterococci from blood samples, 14 E. faecium, 10 E. fecalis and one E. gallinarum were identified. In 2005, Mohanty et al. identified 24 E. faecium, 7 E. fecalis and one E. gallinarum from 38 enterococci strains [14]. In 2019, Karna et al. characterized four E. faecium and one E. fecalis in five enterococci isolates [15]. In another study, Rahbar et al. (2016) isolated Enterococcus spp. from various wards of a university hospital in Tehran, Iran [16]. They identified 74 E. faecalis and 46 E. faecium from a total number of 120 enterococci isolates. In the current study, genetic identification of the enterococcal isolates was carried out using a pair of primers for enterococcal tuf gene and Sanger partial sequencing method. Previously, Li et al. (2012) used the tuf gene as an appropriate target for the identification of Gram-positive cocci [17]. Furthermore, E. faecium is a gut commensal of humans and animals but also enlisted in global priority list of multidrugresistant pathogens by the World Health Organization (WHO) [18]. In the current study, isolated E. faecium strains were resistant to vancomycin, erythromycin, clindamycin, ceftriaxone and cefoxitine. Based on the published studies, 70% of the isolated E. faecium strains from hospitals in Tehran were resistant to vancomycin and erythromycin [19]. In a similar study on clinical samples by Rahbar et al. (2016) in Tehran, antibiotic resistance of enterococcal isolates to erythromycin was reported as 79% and to vancomycin as 51% [16].

In the present study, three various types of phages were isolated from a clinical strain of E. faecium using various wastewater sources. In previous studies, phages have been isolated from various wastewater sources such as raw, hospital and municipal wastewaters [20-22]. In the present study, all the isolated phages included lytic phages due to the clear plaques on agar plates [23]. Overall, three various phages were isolated on E. faecium, namely as EntfacYE1–3. These bacteriophages respectively belonged to Myoviridae, Siphoviridae and Inoviridae families of the Caudovirales order. Based on the literatures, these phages are mostly isolated on E. faecalis and E. faecium [24]. In a study in 2020, a Siphoviridae phage was isolated on E. fecalis, which was previously identified in 2012 [25, 26]. In 2019 Wandro et al. carried out a study on coevolution of E. faecium isolates from healthy human stools in absence and presence of Myoviridae phages [27]. In the current study, specificity assessment of the isolated phages demonstrated that the phages could lyse Streptococcus spp. as well as the Enterococcus spp. In fact, this broader host-specify of the isolated phages can be important due to the horizontal transfer of antimicrobial resistance genes and other virulence genes between various bacterial genera [28]. Moreover, this may be significant regarding treatment of a wider spectrum of the bacterial pathogens using a single phage, instead of a phage cocktail. Further characterization of the bacterial responses to phage infections is important for understanding how phages modulate bacterial physiology, which opens new horizons toward effective phage therapies against multiple-drug resistant bacteria [29]. Phage therapy, a promising alternative to antimicrobial treatment of bacterial diseases, is becoming further popular, especially due to the rapid spread of antimicrobial resistance in bacteria and recent restrictions on antimicrobial uses [16].


Bacteriophages are spread in the environment, especially in wastewaters. Nowadays, clinically-isolated bacteria show multiple resistance to antibiotics, which have become serious problems for the medical specialists. Alternatively, phages can be used to solve these problems. Therefore, further clinical studies are necessary to investigate therapeutic properties of the phages on bacterial multipleresistant strains.

Conflict of Interest

The authors declare no conflict of interest.


The authors thank staff within the Microbiology Laboratory for their kind helps. The current study was financially supported by a grant from the Deputy Dean for Research, Tehran University of Medical Sciences (grant no. 97-02-27-39571).


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