Skip to main content
Download PDF

In vitro evaluation of two novel Escherichia bacteriophages against multiple drug resistant avian pathogenic Escherichia coli

BMC Infectious Diseases volume 24, Article number: 497 (2024) Cite this article

Abstract

Background

In recent years, there has been a growing interest in phage therapy as an effective therapeutic tool against colibacillosis caused by avian pathogenic Escherichia coli (APEC) which resulted from the increasing number of multidrug resistant (MDR) APEC strains.

Methods

In the present study, we reported the characterization of a new lytic bacteriophage (Escherichia phage AG- MK-2022. Basu) isolated from poultry slaughterhouse wastewater. In addition, the in vitro bacteriolytic activity of the newly isolated phage (Escherichia phage AG- MK-2022. Basu) and the Escherichia phage VaT-2019a isolate PE17 (GenBank: MK353636.1) were assessed against MDR- APEC strains (n = 100) isolated from broiler chickens with clinical signs of colibacillosis.

Results

Escherichia phage AG- MK-2022. Basu belongs to the Myoviridae family and exhibits a broad host range. Furthermore, the phage showed stability under a wide range of temperatures, pH values and different concentrations of NaCl. Genome analysis of the Escherichia phage AG- MK-2022. Basu revealed that the phage possesses no antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and any E. coli virulence associated genes. In vitro bacterial challenge tests demonstrated that two phages, the Escherichia phage VaT-2019a isolate PE17 and the Escherichia phage AG- MK-2022. Basu exhibited high bactericidal activity against APEC strains and lysed 95% of the tested APEC strains.

Conclusions

The current study findings indicate that both phages could be suggested as safe biocontrol agents and alternatives to antibiotics for controlling MDR-APEC strains isolated from broilers.

Peer Review reports

Background

Avian collibasilosis is one of the most important causes of morbidity, mortality, and considerable economic loss in the poultry industry worldwide [1, 2]. This complex syndrome is characterized by colisepticemia, coligranuloma, pleuropneumonia, salpingitis, omphalitis, pericarditis, peritonitis, airsacculitis, perihepatitis, and swollen head syndrome in chickens [2], and osteomyelitis complex in turkeys [3]. Avian pathogenic Escherichia coli (APEC) is a subdivision of extraintestinal pathogenic E. coli (ExPEC), known as the main etiological agent of avian collibasiosis [2, 4]. The APEC strains have also been reported as potential zoonotic pathogens [5, 6].

Over the past decades, antibiotics have traditionally been used for the treatment and control of bacterial infections; however, the long-term and excessive use of antibiotics has led to an increase in antimicrobial resistance (AMR), and subsequently, bacteria show high multidrug resistance (MDR) properties, which poses a serious concern for animal, human and public health [7,8,9]. To combat these emerging MDR bacteria, novel strategies and alternative treatments, such as phage therapy (using bacteriophages to target and kill specific bacteria), are urgently needed [10,11,12]. Recently, the biocontrol of bacterial pathogens using bacteriophages (short phages) has attracted growing interest as an alternative to conventional antibiotic therapies, particularly against MDR bacterial pathogens [13,14,15,16]. There are 1031 bacteriophage particles in the biosphere, which are the most abundant on Earth [17, 18]. Phage therapy has several potential advantages compared to traditional antibiotic therapy. Some of these advantages include the following circumstances: (i) highly specific targeted; phages can be engineered to target specific bacteria, which means that they can be more effective in eliminating the specific pathogen causing an infection while leaving other bacteria in the body intact; (ii) a lower risk of resistance; bacteria can develop resistance to antibiotics over time, however because phages target specific bacteria, the risk of resistance development is lower; (iii) fewer side effects can occur; antibiotics can sometimes cause side effects such as stomach upset, diarrhea, or allergic reactions, however, phages are typically less likely to cause side effects, and (iv) availability; phages are naturally occurring viruses that can be found in soil, water, and other environments, which means that they are widely available and relatively easy to isolate [11, 19,20,21].

In recent years, avian collibasilosis prevalence, molecular characterization and MDR properties of APEC strains have been reported in Iran [7, 22,23,24], and considering this problem, applying new effective treatments for MDR- APEC strains is necessary. Therefore, the aims of this study were to (1) isolate and check the stability and characterize the physical and genetic properties of a new E. coli-specific phage (Escherichia phage AG- MK-2022. Basu), (2) determination of the phage host range and efficiency of plating of newly isolated Escherichia phage, and (3) evaluation of the bacteriolytic potential of the newly isolated phage (Escherichia phage AG- MK-2022. Basu) and the Escherichia phage VaT-2019a isolate PE17 (GenBank: MK353636.1), which was obtained from a recently published study [25], against MDR APEC strains, in vitro.

Methods

Bacterial strains

In this study, seven ATCC bacteria prepared from the strain collection of the Faculty of Veterinary Medicine (Bu-Ali Sina University, Hamedan, Iran) were used as reference strains (Table 1). In addition, a total of 100 APEC isolates obtained from previous research [24] were used for the studies. All the strains were revived by subculture in Tryptic Soy Broth (TSB; Merck, Germany) at 37 °C for 24 to 48 h separately.

Table 1 Bacterial strains used for the host range determination of the Escherichia phage AG- MK-2022. Basu
Full size table

Bacteriophage isolation, purification, and propagation

In this study, two bacteriophages, Escherichia phage VaT-2019a isolate PE17 and Escherichia phage AG- MK-2022. Basu, were used. Escherichia phage VaT-2019a isolate PE17 (GenBank: MK353636.1) was obtained from a recently published study [25]. The second phage, Escherichia phage AG- MK-2022. Basu was isolated and purified from sewage water collected from a poultry slaughterhouse in Hamedan, Iran, using a modified method as described previously [26]. Briefly, the sewage water samples collected from different parts of the poultry slaughterhouse were held at 4 °C for 24 h to allow large particles to sediment. Then, 100 ml of each sample was centrifuged (6000 rpm, 15 min, 4 °C), and the supernatant was passed through a 0.22 μm syringe filter (FilterBio® Sterile syringe filters, Nantong FilterBio Membrane Co, China). Subsequently, 100 µl of each filtered sample was mixed with 3 ml of E. coli ATCC25922 overnight culture supplemented with 10 mM MgSO4 and incubated at 37 °C at 160 rpm for approximately 5 h. Finally, the mixture was centrifuged at 6000 rpm for 10 min to remove bacterial cells, and the supernatant was filtered again. The filtered suspension was tested for the presence of bacteriophage using a double layer agar assay (DLA) as described previously [27]. The isolated phage was purified by picking a single plaque (lack of bacterial growth) from the plate, transferring it to TSB containing host bacteria, incubating it at 37 °C for 24 h, and then centrifuging (600 rpm, 10 min). The supernatant was collected and used as a source of isolated phage. The procedure was repeated three times to obtain pure phage cultures, and the purified phage was stored at 4 °C until use [28].

Bacteriophage titer measurement

Titers of phage suspensions were then measured using the double-layer agar (DLA) technique as was previously described [27, 29]. Briefly, the phage suspensions were diluted (10− 1, 10− 9) using SM buffer (8 mM MgSO4⋅7H2O, 50 mM Tris–HCl, 100 mM NaCl) and 100 µl of each phage dilution plus 200 µl of host bacteria mixed with 3 ml melted 0/5% brain heart infusion (BHI) agar (Merck Millipore, Germany). The mixture was then poured onto solidified plates containing 1/5% BHI agar medium as a surface layer and incubated at 37 °C for 24 h. The number of lysed plaques was counted, and the result are reported as plaque forming units per milliliter (PFU/ml).

Electron microscopy

Transmission electron micrographs were obtained according to a previously described procedure [30]. Briefly, a highly concentrated phage suspension (109 PFU/mL) was centrifuged at 30,000 ×g for 3 h at 4 °C, the supernatant was poured off, the pellet was resuspended in 0.1 M ammonium acetate solution, and then the suspension was filtered (0.22 mm). Transmission electron microscopy analysis of the samples was performed by Transmission Electron Microscope (Zeiss, Germany) in the Laboratory of Electron Microscopy, Pasteur Institute, Tehran, Iran.

Phage host range determination and efficiency of plating

The host range of the phage was determined by the spot method [31] using the bacteria listed in Table 1. Overnight cultures (100 µl) of each tested strain were inoculated on the surface of BHI agar plates, the plates were allowed to dry at room temperature, and then 10 µl of phage (109 PFU/ml) was spotted in triplicate onto the surface of the plates. The plates were incubated at 37 °C and monitored for clearance zones. The appearance of clear single plaques was considered evidence of susceptibility to the phage. The efficiency of plating (EOP) is a measurement of the bactericidal efficiency of the bacteriophage on a given bacterial cell line compared to the host bacteria [31]. The EOP was determined for positive bacteria in the spot test using the DLA method [27]. The experiments were performed thrice for each strain. The EOP was calculated based on (average PFU on target bacteria/average PFU on host bacteria) [31]. The results were classified based on EOP values as high, moderate, low, or insufficient. (EOP ≥ 0.5, high efficiency; 0.1 ≤ EOP < 0.5, moderate efficiency; 0.001 < EOP).

Phage stability

The stability of phage under different conditions, including temperature, pH, and NaCl, was evaluated using previously described methods [32, 33], with few modifications. To test the temperature stability of Escherichia phage AG- MK-2022. Basu. 10 µl of phage suspension (109 PFU/mL) was incubated at temperatures ranging from 4 °C to 80 °C for 1 h, followed by determining phage titer and viability using the DLA method. The phage stability at different pH values was evaluated by mixing 10 µl of concentrated phage suspension (109 PFU/mL) with 990 µl of SM buffer and adjusting at various pH values ranging from 1 to 14. The mixture was then incubated at 37 °C for 1 h, and then the phage titer was determined using the DLA method. The stability of the phage was tested against various concentrations of NaCl by incubating phage (109 PFU/mL) with varying ratios of NaCl (1 to 11%) at 37 °C for 60 min, and the phage titer was assayed using the DLA method.

Phage adsorption assay

To determine Phage adsorption assay of Escherichia phage AG- MK-2022. Basu, 1 ml of fresh host bacterial culture of E. coli ATCC 25,922 (10 9 CFU/ml) was mixed with 10 µl of phage suspension (108 PFU/ml) to reach a multiplicity of infection (MOI) = 0.1 and, then pre-warmed fresh TSB was added to mixture. The phage-host mixture incubated with shaking at 160 rpm and 37 °C. After 0, 5, and 10 min, 100 µl of suspension were collected, and added in 900 µl SM buffer, centrifuged at 6,000 g for 1 min and supernatants were filtered by using 0.2 μm membrane filter. The filtered liquid was cultured using the DLA method, and the titer of the nonadsorbed phages was calculated. The percentage of nonadsorbed phages at every given time was determined by dividing the phage titer at 5 and 10 min by the phage titer at time zero [34, 35].

One-step growth curve

A one-step growth experiment was performed as described previously [32], with some modifications. In brief, a 1 ml log phase of the E. coli ATCC 25,922 (1.5 × 108 CFU/mL) and, then a 1ml Escherichia phage AG- MK-2022. Basu suspension (106 PFU/mL) were added to the host at an MOI = 0.1 and to allow phage adsorption to bacterial cells for 5 min at room temperature. The phage-bacterial mixture was centrifuged at 8000 × g for 5 min and then the supernatant was discarded. After three washes, the pellet was resuspended in 10 ml of pre-warmed fresh TSB and incubated with shaking at 180 rpm and 37 °C. Subsequently, 100 µl of the mixture were collected every 10 min, mixed with 900 µl SM buffer and centrifuged at 8500 × g for 1 min. The supernatant was filtered by a 0.22 μm filter, and the phage titer was measured by DLA method. The burst size was calculated as the ratio of the final number of phage particles to the initial number of infected host cells at the beginning of the test.

Phage genome extraction and analysis

In this study, we used the randomly amplified polymorphic DNA (RAPD)-PCR technique described previously [36] for rapid screening and typing of Escherichia phage AG- MK-2022.Basu. Before the RAPD-PCR assay, genomic DNA was extracted from purified phage at the highest concentration using a phage DNA isolation kit (Norgen Biotek Corp., Thorold, ON, Canada) following the manufacturer’s instructions. The extracted DNA purity and concentration were assayed by gel electrophoresis and a NanoDrop spectrometer (Thermo Scientific™ NanoDrop 2000, Waltham, MA, USA). RAPD-PCR amplification was performed using P1 (5′-CCGCAGCCAA-3′) and P2 (5′-AACGGGCAGA-3′) primers [36]. Master mix preparation and thermal conditions were done according to the previously described protocol [25] in the SimpliAmp™ thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). In each run, Escherichia phage VaT-2019a isolate PE17 and distilled deionized water were used as positive and negative controls. In addition, extracted phage DNA was investigated for the presence of the E. coli virulence associated genes (VAGs) stx1, stx2, and hylA, using the multiplex PCR method, as described previously [24]. Oligonucleotides sequences for VAGs detection were from previously published study [37]. Primers sequences, expected amplicon sizes, and thermal cycling conditions are presented in supplementary Table S1. E. coli serotype O157: H7 ATCC 43,895 harboring stx1, stx2 and hylA genes were used as positive controls for the PCR. PCR-amplified products were analyzed in 0.8% (w/v) agarose gel (SinaClon, Iran) and electrophoresed at 110 V for 55 min. The gels were visualized under UV light and photographed using a UV Imager (Transilluminator, Vilber Lourmat, France). Moreover, the presence of mobile genetics elements (MGEs) i.e. class 1 (intI1) and class 2 (intI2) integrons, and antibiotic resistance genes (ARGs) associated with resistance to β-lactams (blaTEM), tetracycline (tetA), plasmid-mediated quinolone (qnrA), sulfonamide (sul1), and trimethoprim(dfrA1) were assessed in Escherichia phage AG- MK-2022.Basu using the PCR method. The primers used, and thermal cycling conditions are given in supplementary Table S1. The previously described procedure was used for master mix preparation and gel electrophoresis [7].

Phage bacteriolytic activity

In vitro bacteriolytic activity of Escherichia phage AG- MK-2022. Basu was determined using the bacterial growth reduction assay method as previously described [38,39,40]. In brief, an overnight culture of E. coli ATCC 25,922 was inoculated in BHI broth and incubated at 37 °C with shaking at 180 rpm until the optical density at 600 nm (OD600 nm) reached 1 (early exponential growth phase). Then, 200 µl of bacterial culture was mixed with 100 µl of phage stock solution (109 PFU/mL), and this mixture was incubated at 37 °C for 24 h. The samples were collected after 0, 2, 4, 6, 8, 10, 12 and 24 h of incubation, and bacterial growth was monitored by measurement of OD 600 nm using a Shimadzu (Kyoto, Japan) mini UV 1240 spectrophotometer. All assays were performed in triplicate, and in each experiment, a bacterial control (BC) and a phage control (PC) were used. The bacterial control was inoculated only with bacteria, and the phage control was inoculated with phages only.

Phage bactericidal activity against avian pathogenic E. Coli

In vitro bactericidal potential of both phages (Escherichia phage VaT-2019a isolate PE17 and Escherichia phage AG- MK-2022. Basu) against APEC strains (n = 100) were estimated by double agar overlay plaque assay [41], spot method, and bacterial growth reduction assay procedure as described above. During these experiments, the titer of phage was 109 PFU/ml, and APEC strains at exponential growth phase (OD 600 nm = 1) were used instead of the host bacteria. The APEC strains were isolated from broiler chickens with clinical signs of colibacillosis on eight different broiler farms in Hamedan, western Iran. The sampling details, bacterial isolation and identification methods, antibiotic resistance profile, and MDR properties of these strains were described earlier [7, 24].

Statistical analysis

Independent t-test was used to compare bacterial growth reduction rate (section Phage bactericidal activity against avian pathogenic E. coli) in the presence and absence of the phages. In addition, statistical analysis of the results of the bacteriophages infection test were performed by independent t-test and, the mean logarithm of the number of bacteria in the two groups. The IBM SPSS Statistics software version 26 programs for Windows (IBM Corp, Armonk, NY) was used for statistical analysis.

Results

Phage isolation and purification

Escherichia phage AG- MK-2022. Basu was isolated from poultry slaughterhouse sewage water samples in Hamedan, Iran, using E. coli ATCC 25,922 as a host. This phage formed large, clear plaques specifically in the presence of a host strain with a diameter of 1–4 millimeters (Fig. 1). Here, we describe the characterization of this bacteriophage.

Fig. 1
figure 1

The plaques of Escherichia phage AG- MK-2022.Basu formed on a double-layered agar plate. A: 109PFU/mL. B: 103 PFU/mL

Full size image

Bacteriophage morphology

Transmission electron microscopy (TEM) revealed that Escherichia phage AG- MK-2022. Basu had a tail (caudovirales) and structural characteristics similar to phages of the family Myoviridae (Fig. 2).

Fig. 2
figure 2

Transmission electron microscopy (TEM) of the Escherichia phage AG- MK-2022.Basu. A: Scale bar, 100 nm (Magnification = X50000) and B: Scale bar, 60 nm (Magnification = X85000)

Full size image

Phage host range and EOP

The host range of Escherichia phage AG- MK-2022. Basu was estimated by spot test. Four out of seven tested strains (57.1%) showed a clear plaque in a spot assay test, including two E. coli strains, Salmonella and Pseudomonas aeruginosa (Table 1).

The EOP assay was performed for positive bacteria in the spot test, and the results of the EOP test indicate that the Escherichia phage AG- MK-2022. Basu showed high, moderate, and low efficiency in 1, 2 and 1 strains, respectively (Table 2).

Table 2 Host range and efficiency of plating (EOP) of the Escherichia phage AG- MK-2022. Basu
Full size table

Phage stability

The results of Escherichia phage AG- MK-2022. Basu stability under various conditions, including temperature, pH, and NaCl concentration, is demonstrated in Fig. 3. The heat stability results revealed that the Escherichia phage AG- MK-2022. Basu survived at 4 °C to 80 °C, and there was no significant reduction in phage titer after incubation at temperatures between 4 °C and 40 °C, but the phage titer dropped significantly to 4.6 log PFU/ml at 60 °C and to 2.9 log PFU/ml at 80 °C compared to 4 °C. However, the phage could survive even incubation at 80 °C, but the lytic activity of phage was significantly reduced. Additionally, the phage remained stable when stored at 4 °C for four months (Fig. 3-A). As shown in Fig. 3-B, there was no significant difference in phage titer after 1 h incubation at pH 4–10. None of the phages survived at pH 2; however, at pH = 3 and 12, a decrease in phage titer was observed compared to standard conditions (pH = 7). There was no significant reduction in phage titer in the 1–9% NaCl range; however, increasing the NaCl concentration to 11% and 13% caused a significant drop in phage titer by 0.73 and 0.88 log PFU/ml, respectively, compared to the 1% concentration of NaCl (Fig. 3-C).

Fig. 3
figure 3

Escherichia phage AG- MK-2022. Basu stability in various conditions: (A) temperature; (B) pH; (C) NaCl

Full size image

Phage adsorption assay and one-step growth curve

Phage adsorption assay showed that approximately 99.2% of the Escherichia phage AG- MK-2022. Basu particles adsorbed to the host bacterial cells after 5 min (Fig. 4-A). One-step growth curve revealed that Escherichia phage AG- MK-2022. Basu had a latent period of 10 min and a burst size of 152 PFU / host cell (Fig. 4-B).

Fig. 4
figure 4

Adsorption of Escherichia phage AG- MK-2022. Basu to E. coli ATCC 25,922 (A); One-step growth curves of Escherichia phage AG- MK-2022. Basu in the presence of E. coli ATCC 25,922 as host (B)

Full size image

Genome analysis

As shown in Fig. 5-a, RAPD band patterns with amplicon sizes in the range of 1000–1500 bp with primer P1 and two distinct band patterns with amplicons ranging in size from 400 to 500 bp and 700–800 bp with primer P2 were observed after gel electrophoresis. Pure DNA of Escherichia phage VaT-2019a isolate PE17 was used as a control (Fig. 5-a). By applying RAPD PCR, it is also possible to discriminate between different phage lineages, with no need for whole genome sequencing [18, 36]. The PCR results for VAGs revealed that E. coli virulence associated genes (stx1, stx2, and hylA) were not detected in Escherichia phage AG- MK-2022. Basu (Fig. 5-b and 5-c). The PCR results for ARGs and MGEs showed that the Escherichia phage AG- MK-2022. Basu does not contain genes encoding antibiotic resistance (blaTEM, tetA, qnrA, sul1, and dfrA1), and integrons (intI1 and intI2).

Fig. 5
figure 5

(a) RAPD band patterns obtained from Escherichia phage VaT-2019a isolate PE17(B1 and B2) and, Escherichia phage AG- MK-2022.Basu (K1 and K2) using P1 and P2 primers, respectively; (b) and (c) PCR for detection of Escherichia coli virulence associated genes stx1, stx2, and hylA. (L) marker 100 bp, (N) negative control, (P) positive control (Escherichia coli O157: H7 ATCC 43,895), (B1) Escherichia phage VaT-2019a isolate PE17 with primer P1, (K1) Escherichia phage AG- MK-2022.Basu with primer P1, (B2) Escherichia phage VaT-2019a isolate PE17 with primer P2, (K2) Escherichia phage AG- MK-2022.Basu with primer P2

Full size image

Phage bacteriolytic activity

A bacterial growth reduction assay was used to evaluate the lytic activity of Escherichia phage AG- MK-2022. Basu against the host strain (E. coli ATCC25922) in vitro. The results revealed that Escherichia phage AG- MK-2022. Basu exhibited strong lytic activity against their original host (E. coli ATCC25922) in vitro, and significant decreases in the viability of bacterial strains were observed after phage administration (MOI = 1) compared to the bacterial control (Fig. 6).

Fig. 6
figure 6

Bacterial challenge test of phage Escherichia phage AG- MK-2022. with Escherichia. coli ATCC 25,922. Bac + phage: bacteria + phage, bac: bacteria

Full size image

Phage bactericidal activity against avian pathogenic E. Coli

Tables S1 and S2 describes the in vitro bacteriolytic activity of both phages (Escherichia phage VaT-2019a isolate PE17 and Escherichia phage AG- MK-2022. Basu) against APEC strains (n = 100) by double agar overlay plaque assay, spot test, and bacterial growth reduction assay. As demonstrated in Table S2, Escherichia phage AG- MK-2022 and Escherichia phage VaT-2019a isolate PE17 lysed 95 (95%) of the 100 APEC strains in the spot assay test (Table S2). In addition, the in vitro bacterial growth reduction assay results revealed that both phages cause significant decreases in the viability of 95% of the examined APEC strains (OD 600 nm) six hours after phage addition compared with the control group (Fig. 7 and Tables S1 and S2). The double agar overlay plaque assay results indicate that most APEC strains (45%) were susceptible to phage dilution at 10− 1 (Table 3).

Fig. 7
figure 7

Bacteriolytic activity of Escherichia phage AG- MK-2022. Basu and, Escherichia phage VaT-2019a isolate PE17 against Avian Pathogenic Escherichia coli (APEC) strains in vitro

Full size image
Table 3 Phages bactericidal activity against avian pathogenic Escherichia coli (APEC) in different dilutions of phages
Full size table

Statistical analysis results

A significant decrease was observed for the OD values at 600 nm in 95 / 100 of tested APEC strains in the presence of phages compared to the mean values in the absence of the phages (Table S2).

Discussion

Recently, the emergence of multidrug-resistant bacterial pathogens highlights the need for new strategies to combat them and has demonstrated the value of phages as antibacterial agents for medical and veterinary applications [17, 32]. In addition, MDR-APEC strains pose a significant threat to poultry health and lead to economic losses in the poultry industry [7, 22, 42,43,44], emphasizing the need for alternative approaches for decreasing the incidence of MDR-APEC strains in poultry [45].

In this study, the new phage (Escherichia phage AG- MK-2022. Basu) was isolated from poultry slaughterhouse sewage and selected because it has high host specificity to E. coli strains. Several studies have reported the isolation of lytic specific phages for E. coli from poultry sewage samples, and these findings confirm that sewage samples are rich sources for isolating lytic bacteriophages [35, 46, 47].

The morphological characteristics of the isolated phage observed by transmission electronic microscopy (TEM) indicate that Escherichia phage AG- MK-2022. Basu belong to the order Caudovirales and family Myoviridae. The same phage types have been isolated from sewage and morphologically characterized previously [25, 47, 48].

Host range determination findings revealed that the Escherichia phage AG- MK-2022. Basu not only lysed the host bacterium but also showed lytic activity against four out of the seven tested bacteria, which were all Gram-negative bacteria, so this phage is a broad-host-range phage with the ability to infect three different genera (Escherichia, Salmonella and Pseudomonas) and may have a polyvalent nature. Previous studies have also reported the isolation and identification of polyvalent Escherichia phages that are able to lyse wide ranges of Gram-negative bacteria simultaneously [25, 28, 49,50,51,52]. However, the results of the EOP test indicated that Escherichia phage AG- MK-2022. Basu was highly specific and effective against E. coli strains. Similar findings were reported in earlier studies that introduced broad host range Escherichia phages that were extremely effective against E. coli strains [48, 53, 54].

Bacteriophage survival under different conditions is one of the most important criteria in the selection of suitable phages to be used in phage therapy [28]. Escherichia phage AG- MK-2022. Basu exhibited stability at a wide range of temperatures (4–80 °C), pH values (4–10), and NaCl concentrations (1–13%). These findings are in accordance with prior investigations [33, 55], which have isolated and identified highly stable Escherichia phages. The stability of phage in these conditions indicates that this phage could be used in a variety of environmental conditions.

In the current study, genome analysis of the Escherichia phage AG- MK-2022. Basu revealed that the isolated phage genome possesses no antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and E. coli virulence associated genes (VAGs) assessed. These findings was in accordance with previous studies [25, 30, 54, 56] which reported that Escherichia phage genome did not harbor any genes associated with pathogenicity and distribution of antibiotic resistance. These findings indicate that the phage may be safely used as a biocontrol agent, however more work, such as whole genome sequencing of the isolated phage, is necessary to achieved more information regarding its safe use.

In recent years, there has been growing interest in phage therapy as an effective therapeutic tool against colibacillosis caused by APEC due to the increasing number of multidrug-resistant APEC strains [54, 57, 58]. The in vitro bacterial challenge tests demonstrated that two phages, Escherichia phage VaT-2019a isolate PE17 and Escherichia phage AG- MK-2022. Basu was highly effective and exhibited high bactericidal activity against the APEC strains we tested. Furthermore, these phages could be used as an alternative to antibiotics for controlling MDR-APEC strains isolated from broilers. Previous studies reported the characterization and application of bacteriophages against APEC strains of poultry origin. A very recent study from Pakistan, Sattar et al. [40]. reported the isolation, characterization, and genome analysis of two lytic phages (Escherichia phage SKA49 and Escherichia phage SKA64) against the MDR strain of APEC and stated that these phages can be good candidates for the control of APEC strains. Zhang et al. [59] introduced phage Bp7 as a wide host range phage and suggested its polyvalent nature and as an alternative to antimicrobials for controlling drug-resistant E. coli in chickens [59]. Tang et al. [54] isolated a newly lytic phage, CE1, from broiler feces. Evaluation of the bactericidal activity of this phage against APEC strains in vitro and in vivo confirmed that the phage has a broad host range and lysed 56.9% (33/58) of highly pathogenic strains of APEC. Nicolas et al. [60] isolated and characterized 19 novel phage collections against APEC strains and reported a cocktail of eight phages to be a promising candidate for the biocontrol of avian colibacillosis. In another study, Kazibwe et al. [45] described seven lytic bacteriophages against APEC and reported that two phages, UPEC04 and UPEC10, had combined lytic activity against APEC strains isolated from broilers. A study performed by Zhou et al. [48] isolated and described JS09 phage from sewage, which was able to infect some clinically isolated antibiotic-resistant APEC and ETEC strains and suggested the phage as a candidate for phage therapy to control colibacillosis in animals.

There were some limitations in the present study such as limitation in whole genome sequencing of newly isolated phage (Escherichia phage AG- MK-2022. Basu) because of resources. Moreover, whole genome sequencing could provide insightful information about the newly isolated phage regarding its safe use.

Conclusion

In conclusion, this study confirmed two novel phages, the Escherichia phage VaT-2019a isolate PE17 and the Escherichia phage AG- MK-2022. Basu exhibited strong antimicrobial effects against APEC strains in vitro and could be suggested as novel biocontrol agents to combat the occurrence of MDR-APEC strains. In vivo evaluations will be suggested to achieve a better picture of probable therapeutic/biocontrol applications of these phages in poultry farms.

Data availability

The datasets generated and analysed during the current study are available in the NCBI—GenBank—Nucleotide platform (https://www.ncbi.nlm.nih.gov/genbank/) and can be accessed through accession number: GenBank: MK353636.1.

References

  1. Ewers C, Janssen T, Kiessling S, Philipp HC, Wieler LH. Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry. Vet Microbiol. 2004;104(1–2):91–101.

    Article  CAS  PubMed  Google Scholar 

  2. Nolan LK, Vaillancourt J-P, Barbieri NL, Logue CM. Colibacillosis. In: Diseases of Poultry edn.; 2020: 770–830.

  3. Huff GR, Huff WE, Rath NC, Balog JM. Turkey osteomyelitis complex. Poult Sci. 2000;79(7):1050–6.

    Article  CAS  PubMed  Google Scholar 

  4. Bonnet C, Diarrassouba F, Brousseau R, Masson L, Topp E, Diarra MS. Pathotype and antibiotic resistance gene distributions of Escherichia coli isolates from broiler chickens raised on antimicrobial-supplemented diets. Appl Environ Microbiol. 2009;75(22):6955–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Tonini da Rocha D, De Oliveira Salle F, Apellanis Borges K, Quedi Furian T, Pinheiro do Nascimento V, Luiz, de Souza Moraes H. Tadeu Pippi Salle C: Avian pathogenic Escherichia coli (APEC) and uropathogenic Escherichia coli (UPEC): characterization and comparison. J Infect Dev Ctries 2021; 15(7):962–971.

  6. Stromberg ZR, Johnson JR, Fairbrother JM, Kilbourne J, Van Goor A, Curtiss RR, Mellata M. Evaluation of Escherichia coli isolates from healthy chickens to determine their potential risk to poultry and human health. PLoS ONE. 2017;12(7):e0180599.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Goudarztalejerdi A, Mohammadzadeh A, Niazi K, Mohammad Mirzaei M. High prevalence of multidrug resistance and biofilm-formation ability among avian Escherichia coli isolated from broilers in Iran. Microb Drug Resist. 2022;28(2):244–54.

    Article  CAS  PubMed  Google Scholar 

  8. Schroeder CM, Zhao C, DebRoy C, Torcolini J, Zhao S, White DG, Wagner DD, McDermott PF, Walker RD, Meng J. Antimicrobial resistance of Escherichia coli O157 isolated from humans, cattle, swine, and food. Appl Environ Microbiol. 2002;68(2):576–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Vivas R, Barbosa AAT, Dolabela SS, Jain S. Multidrug-resistant bacteria and alternative methods to control them: an overview. Microb Drug Resist. 2019;25(6):890–908.

    Article  CAS  PubMed  Google Scholar 

  10. Medina E, Pieper DH. Tackling threats and future problems of multidrug-resistant bacteria. In: How to Overcome the Antibiotic Crisis: Facts, Challenges, Technologies and Future Perspectives edn. Edited by Stadler M, Dersch P. Cham: Springer International Publishing; 2016: 3–33.

  11. Abuladze T, Li M, Menetrez MY, Dean T, Senecal A, Sulakvelidze A. Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach, broccoli, and ground beef by Escherichia coli O157:H7. Appl Environ Microbiol. 2008;74(20):6230–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Necel A, Bloch S, Nejman-Faleńczyk B, Grabski M, Topka G, Dydecka A, Kosznik-Kwaśnicka K, Grabowski Ł, Jurczak-Kurek A, Wołkowicz T, et al. Characterization of a bacteriophage, vB_Eco4M-7, that effectively infects many Escherichia coli O157 strains. Sci Rep. 2020;10(1):3743.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. García P, Martínez B, Obeso JM, Rodríguez A. Bacteriophages and their application in food safety. Lett Appl Microbiol. 2008;47(6):479–85.

    Article  PubMed  Google Scholar 

  14. Golkar Z, Bagasra O, Pace DG. Bacteriophage therapy: a potential solution for the antibiotic resistance crisis. J Infect Dev Ctries. 2014;8(2):129–36.

    Article  PubMed  Google Scholar 

  15. Housby JN, Mann NH. Phage therapy. Drug Discov Today. 2009;14(11–12):536–40.

    Article  CAS  PubMed  Google Scholar 

  16. Shasha SM, Sharon N, Inbar M. Bacteriophages as antibacterial agents. Harefuah. 2004;143(2):121–5.

    PubMed  Google Scholar 

  17. O’Flaherty S, Ross RP, Coffey A. Bacteriophage and their lysins for elimination of infectious bacteria. FEMS Microbiol Rev. 2009;33(4):801–19.

    Article  PubMed  Google Scholar 

  18. Ács N, Gambino M, Brøndsted L. Bacteriophage enumeration and detection methods. Front Microbiol. 2020;11:594868.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Cairns BJ, Timms AR, Jansen VA, Connerton IF, Payne RJ. Quantitative models of in vitro bacteriophage-host dynamics and their application to phage therapy. PLoS Pathog. 2009;5(1):e1000253.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Kazi M, Annapure US. Bacteriophage biocontrol of foodborne pathogens. J Food Sci Technol. 2016;53(3):1355–62.

    Article  PubMed  Google Scholar 

  21. Loc-Carrillo C, Abedon ST. Pros and cons of phage therapy. Bacteriophage. 2011;1(2):111–4.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Kalantari M, Sharifiyazdi H, Asasi K, Abdi-Hachesoo B. High incidence of multidrug resistance and class 1 and 2 integrons in Escherichia coli isolated from broiler chickens in South of Iran. Vet Res Forum. 2021;12(1):101–7.

    PubMed  PubMed Central  Google Scholar 

  23. Ghorbani A, Khoshbakht R, Kaboosi H, Shirzad-Aski H, Peyravii Ghadikolaii F. Study on antibiotic resistance and phylogenetic comparison of avian-pathogenic Escherichia coli (APEC) and uropathogenic Escherichia coli (UPEC) isolates. Vet Res Forum. 2022;13(4):569–76.

    PubMed  PubMed Central  Google Scholar 

  24. Goudarztalejerdi A, Mohammadzadeh A, Najafi SV, Nargesi F, Joudari S. Serogrouping, phylotyping, and virulence genotyping of commensal and avian pathogenic Escherichia coli isolated from broilers in Hamedan, Iran. Comp Immunol Microbiol Infect Dis. 2020;73:101558.

    Article  PubMed  Google Scholar 

  25. Mozaffari P, Berizi E, Hosseinzadeh S, Derakhshan Z, Taghadosi V, Montaseri Z, Gotz F. Isolation and characterization of E. Coli O157: H7 novel bacteriophage for controlling this food-borne pathogen. Virus Res. 2022;315:198754.

    Article  CAS  PubMed  Google Scholar 

  26. Lee H, Ku HJ, Lee DH, Kim YT, Shin H, Ryu S, Lee JH. Characterization and genomic study of the novel bacteriophage hy01 infecting both Escherichia coli O157:H7 and shigella flexneri: potential as a biocontrol agent in food. PLoS ONE. 2016;11(12):e0168985.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Schuster H, von Bacteriophages MH, Adams. Interscience Publishers, Inc., New York-London. 1959. 1. Angewandte Chemie 1962; 74:164–164.

  28. Pereira C, Moreirinha C, Lewicka M, Almeida P, Clemente C, Romalde JL, Nunes ML, Almeida A. Characterization and in vitro evaluation of new bacteriophages for the biocontrol of Escherichia coli. Virus Res. 2017;227:171–82.

    Article  CAS  PubMed  Google Scholar 

  29. Harada LK, Silva EC, Rossi FP, Cieza B, Oliveira TJ, Pereira C, Tomazetto G, Silva BB, Squina FM, Vila MM, et al. Characterization and in vitro testing of newly isolated lytic bacteriophages for the biocontrol of Pseudomonas aeruginosa. Future Microbiol. 2022;17:111–41.

    Article  CAS  PubMed  Google Scholar 

  30. Hudson JA, Billington C, Cornelius AJ, Wilson T, On SLW, Premaratne A, King NJ. Use of a bacteriophage to inactivate Escherichia coli O157:H7 on beef. Food Microbiol. 2013;36(1):14–21.

    Article  CAS  PubMed  Google Scholar 

  31. Kutter E. Phage host range and efficiency of plating. Methods Mol Biol. 2009;501:141–9.

    Article  CAS  PubMed  Google Scholar 

  32. Wang C, Chen Q, Zhang C, Yang J, Lu Z, Lu F, Bie X. Characterization of a broad host-spectrum virulent Salmonella bacteriophage fmb-p1 and its application on duck meat. Virus Res. 2017;236:14–23.

    Article  CAS  PubMed  Google Scholar 

  33. Sváb D, Falgenhauer L, Rohde M, Chakraborty T, Tóth I. Identification and characterization of new broad host-range rV5-like coliphages C203 and P206 directed against enterobacteria. Infect Genet Evol. 2018;64:254–61.

    Article  PubMed  Google Scholar 

  34. Azari R, Yousefi MH, Taghipour Z, Wagemans J, Lavigne R, Hosseinzadeh S, Mazloomi SM, Vallino M, Khalatbari-Limaki S, Berizi E. Application of the lytic bacteriophage Rostam to control Salmonella enteritidis in eggs. Int J Food Microbiol. 2023;389:110097.

    Article  CAS  PubMed  Google Scholar 

  35. Khalatbari-Limaki S, Hosseinzadeh S, Shekarforoush SS, Berizi E. The morphological and biological characteristics of a virulent PI phage isolated from slaughterhouse sewage in Shiraz, Iran. Iran J Microbiol. 2020;12(6):616–24.

    PubMed  PubMed Central  Google Scholar 

  36. Gutiérrez D, Martín-Platero AM, Rodríguez A, Martínez-Bueno M, García P, Martínez B. Typing of bacteriophages by randomly amplified polymorphic DNA (RAPD)-PCR to assess genetic diversity. FEMS Microbiol Lett. 2011;322(1):90–7.

    Article  PubMed  Google Scholar 

  37. Paton AW, Paton JC. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. Coli hlyA, rfbO111, and rfbO157. J Clin Microbiol. 1998;36(2):598–602.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Wang Z, Zheng P, Ji W, Fu Q, Wang H, Yan Y, Sun J. SLPW: A virulent bacteriophage targeting methicillin-resistant Staphylococcus aureus in vitro and in vivo. Front microbiol 2016, 7.

  39. Amarillas L, Rubí-Rangel L, Chaidez C, González-Robles A, Lightbourn-Rojas L, León-Félix J. Isolation and characterization of phills, a novel phage with potential biocontrol agent against multidrug-resistant Escherichia coli. Front microbiol 2017, 8.

  40. Sattar S, Bailie M, Yaqoob A, Khanum S, Fatima K, Altaf AURB, Ahmed I, Shah STA, Munawar J, Zehra QA, et al. Characterization of two novel lytic bacteriophages having lysis potential against MDR avian pathogenic Escherichia coli strains of zoonotic potential. Sci Rep. 2023;13(1):10043.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP. Enumeration of bacteriophages by double agar overlay plaque assay. Methods Mol Biol. 2009;501:69–76.

    Article  CAS  PubMed  Google Scholar 

  42. Saha O, Hoque MN, Islam OK, Rahaman MM, Sultana M, Hossain MA. Multidrug-resistant avian pathogenic Escherichia coli strains and association of their virulence genes in Bangladesh. Microorganisms 2020, 8(8).

  43. Azam M, Mohsin M, Johnson TJ, Smith EA, Johnson A, Umair M, Saleemi MK, Sajjad Ur R. Genomic landscape of multi-drug resistant avian pathogenic Escherichia coli recovered from broilers. Vet Microbiol. 2020;247:108766.

    Article  CAS  PubMed  Google Scholar 

  44. Kim YB, Yoon MY, Seo KW, Yoon S, Lee YJ. Molecular characterization of multidrug-resistant avian pathogenic Escherichia coli from broiler chickens in Korea. J Appl Poult Res. 2020;29(4):1101–7.

    Article  CAS  Google Scholar 

  45. Kazibwe G, Katami P, Alinaitwe R, Alafi S, Nanteza A, Nakavuma JL. Bacteriophage activity against and characterisation of avian pathogenic Escherichia coli isolated from colibacillosis cases in Uganda. PLoS ONE. 2020;15(12):e0239107.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Nicolas M, Trotereau A, Culot A, Moodley A, Atterbury R, Wagemans J, Lavigne R, Velge P, Schouler C. Isolation and characterization of a novel phage collection against avian-pathogenic Escherichia coli. Microbiol Spectr. 2023;11(3):e04296–04222.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Oliveira A, Sillankorva S, Quinta R, Henriques A, Sereno R, Azeredo J. Isolation and characterization of bacteriophages for avian pathogenic E. Coli strains. J Appl Microbiol. 2009;106(6):1919–27.

    Article  CAS  PubMed  Google Scholar 

  48. Zhou Y, Bao H, Zhang H, Wang R. Isolation and characterization of lytic phage vb_ecom_js09 against clinically isolated antibiotic-resistant avian pathogenic Escherichia coli and enterotoxigenic Escherichia coli. Intervirology. 2015;58(4):218–31.

    Article  CAS  PubMed  Google Scholar 

  49. Liao Y-T, Zhang Y, Salvador A, Ho K-J, Cooley MB, Wu VCH. Characterization of polyvalent Escherichia phage Sa157lw for the biocontrol potential of Salmonella Typhimurium and Escherichia coli O157:H7 on contaminated mung bean seeds. Front Microbiol 2022, 13.

  50. Duc HM, Son HM, Yi HPS, Sato J, Ngan PH, Masuda Y, Honjoh K-i, Miyamoto T. Isolation, characterization and application of a polyvalent phage capable of controlling Salmonella and Escherichia coli O157:H7 in different food matrices. Int Food Res. 2020;131:108977.

    Article  CAS  Google Scholar 

  51. Amarillas L, Rubí-Rangel L, Chaidez C, González-Robles A, Lightbourn-Rojas L, León-Félix J. Isolation and characterization of phills, a novel phage with potential biocontrol agent against multidrug-resistant Escherichia coli. Front Microbiol. 2017;8:1355.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Jensen EC, Schrader HS, Rieland B, Thompson TL, Lee KW, Nickerson KW, Kokjohn TA. Prevalence of broad-host-range lytic bacteriophages of Sphaerotilus natans, Escherichia coli, and Pseudomonas aeruginosa. Appl Environ Microbiol 1998; 64(2):575–580.

  53. Viscardi M, Perugini AG, Auriemma C, Capuano F, Morabito S, Kim KP, Loessner MJ, Iovane G. Isolation and characterisation of two novel coliphages with high potential to control antibiotic-resistant pathogenic Escherichia coli (EHEC and EPEC). Int J Antimicrob Agents. 2008;31(2):152–7.

    Article  CAS  PubMed  Google Scholar 

  54. Tang Z, Tang N, Wang X, Ren H, Zhang C, Zou L, Han L, Guo L, Liu W. Characterization of a lytic Escherichia coli phage CE1 and its potential use in therapy against avian pathogenic Escherichia coli infections. Front Microbiol 2023, 14.

  55. Jurczak-Kurek A, Gąsior T, Nejman-Faleńczyk B, Bloch S, Dydecka A, Topka G, Necel A, Jakubowska-Deredas M, Narajczyk M, Richert M, et al. Biodiversity of bacteriophages: morphological and biological properties of a large group of phages isolated from urban sewage. Sci Rep. 2016;6(1):34338.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Dini C, De Urraza PJ. Isolation and selection of coliphages as potential biocontrol agents of enterohemorrhagic and Shiga toxin-producing E. Coli (EHEC and STEC) in cattle. J Appl Microbiol. 2010;109(3):873–87.

    Article  CAS  PubMed  Google Scholar 

  57. Wernicki A, Nowaczek A, Urban-Chmiel R. Bacteriophage therapy to combat bacterial infections in poultry. Virol J. 2017;14(1):179.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Abd-El Wahab A, Basiouni S, El-Seedi HR, Ahmed MFE, Bielke LR, Hargis B, Tellez-Isaias G, Eisenreich W, Lehnherr H, Kittler S et al. An overview of the use of bacteriophages in the poultry industry: successes, challenges, and possibilities for overcoming breakdowns. Front Microbiol 2023, 14.

  59. Zhang C, Li W, Liu W, Zou L, Yan C, Lu K, Ren H. T4-like phage Bp7, a potential antimicrobial agent for controlling drug-resistant Escherichia coli in chickens. Appl Environ Microbiol. 2013;79(18):5559–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Nicolas M, Trotereau A, Culot A, Moodley A, Atterbury R, Wagemans J, Lavigne R, Velge P, Schouler C. Isolation and characterization of a Novel Phage Collection against Avian-Pathogenic Escherichia coli. Microbiol Spectr. 2023;11(3):e0429622.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Hossein Payami, and Sajad Varmaziar Najafi for assistance in sampling, Mr Rahim Azari, Mr Behrooz Abbasabadi, Ms Sakineh Azami, Ms Mozhdeh Zolfaghari and Zahra Rahimifar for technical assistance, and professor Saeid Hosseinzadeh (Professor of Food Science and Molecular Biology, Department of Food Hygiene and Public Health, School of Veterinary Medicine Shiraz University, Shiraz, Iran) for English revision.

Funding

This work was supported by a grant from the Bu-Ali Sina University, Hamedan, Iran (Grant No. 1451 to AG).

Author information

Authors and Affiliations

  1. Department of Pathobiology, Faculty of Veterinary Medicine, Bu-Ali Sina University, Hamedan, 6517658978, Iran

    Mobina Karami, Ali Goudarztalejerdi & Abdolmajid Mohammadzadeh

  2. Department of Food Hygiene and Quality Control, School of Nutrition and Food Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

    Enayat Berizi

Authors
  1. Mobina Karami
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ali Goudarztalejerdi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Abdolmajid Mohammadzadeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Enayat Berizi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M. K. Investigation, Methodology, Writing - original draft, Visualization. A.G. Writing - review & editing, Supervision, Project administration, Funding acquisition. A.G., A.M., and E. B. supervised the study. A.G. and E. B. revised the article. All authors reviewed the article.

Corresponding author

Correspondence to Ali Goudarztalejerdi.

Ethics declarations

Ethical approval and consent to participate

All experiments were permitted by the members of the ethics committee, Bu-Ali Sina University, Hamedan, Iran (Approval No. 1605524). All methods reported in the present study are in accordance with relevant guidelines and regulations. It is confirmed that informed consent has been obtained from all subjects.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Karami, M., Goudarztalejerdi, A., Mohammadzadeh, A. et al. In vitro evaluation of two novel Escherichia bacteriophages against multiple drug resistant avian pathogenic Escherichia coli. BMC Infect Dis 24, 497 (2024). https://doi.org/10.1186/s12879-024-09402-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12879-024-09402-0

Keywords

BMC Infectious Diseases

ISSN: 1471-2334

Contact us