Identification of E. coli isolates and growth conditions
Morphological testing of the 65 E. coli isolates on MacConkey agar revealed pink colonies (Figure 1) and under microscope appeared as rods. Biochemical tests revealed that the isolates were E. coli. Identification was also confirmed by the Vitek2 system.
Antibiotic susceptibility testing
The sensitivity of the E. coli isolates to different antibiotics was determined by the disc diffusion method, as shown in Table 1. The data showed that 7.7 % (5/65) of the E. coli isolates were resistant to ertapenem, 9.2 % (6/65) of the isolates were resistant to nitrofurantoin, 21.5% (14/65) of the isolates were resistant to chloramphenicol, 47.7 % (31/65) of the isolates were resistant to tetracycline and gentamicin, 53.8% (35/65) of the isolates were resistant to piperacillin-tazobactam, 67.6% (44/65) of the isolates were resistant to trimethoprim-sulfamethoxazole, 70.7% (46/65) of the isolates were levofloxacin resistant, 81.5% (53/65) of the isolates were cefepime resistant, 95.4% (62/65) of the isolates were cefazoline and ceftriaxone resistant, 97% (63/65) of the isolates were resistant to ciprofloxacin, and finally, ceftazidime, ampicillin, and amoxicillin-clavulanate were not effective against 100% (65/65) of the isolates (Table 1). All 65 isolates were MDR.
Table 1. Antibiotic susceptibility of MDR E. coli isolates
Isolates
|
Antimicrobial agents
|
AMP
|
AMC
|
PIT
|
CZ
|
FEP
|
CTR
|
CAZ
|
ETP
|
GMN
|
TE
|
CIP
|
LEV
|
COT
|
CHL
|
NIT
|
E. coli 1
|
|
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E. coli 2
|
|
|
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|
|
E. coli 3*
|
|
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|
E. coli 4
|
|
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E. coli 5
|
|
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E. coli 6
|
|
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E. coli 7
|
|
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E. coli 8
|
|
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E. coli 9
|
|
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E. coli 10
|
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E. coli 11
|
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E. coli 12
|
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E. coli 13
|
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E. coli 14
|
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E. coli 15
|
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E. coli 16
|
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E. coli 17
|
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E. coli 18
|
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E. coli 19
|
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E. coli 20
|
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E. coli 21
|
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E. coli 22
|
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E. coli 23
|
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E. coli 24
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E. coli 25
|
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E. coli 26
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E. coli 27
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E. coli 28
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E. coli 29
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E. coli 30
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E. coli 31
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E. coli 32
|
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E. coli 33
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E. coli 34
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E. coli 35
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E. coli 36
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E. coli 37
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E. coli 38
|
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E. coli 39
|
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E. coli 40
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E. coli 41
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E. coli 42
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E. coli 43
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E. coli 44
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E. coli 45
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E. coli 46
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E. coli 47
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E. coli 48
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E. coli 49
|
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E. coli 50
|
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E. coli 51
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E. coli 52
|
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E. coli 53
|
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E. coli 54
|
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E. coli 55
|
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E. coli 56
|
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E. coli 57
|
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E. coli 58
|
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E. coli 59
|
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E. coli 60
|
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E. coli 61
|
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E. coli 62
|
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E. coli 63
|
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E. coli 64
|
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E. coli 65
|
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|
The MDR E. coli 3* was selected as the host isolate. It was resistant to ampicillin, amoxicillin-clavulanate, piperacillin-tazobactam, cefazoline, cefepime, ceftriaxone, ceftazidime, tetracycline, ciprofloxacin, levofloxacin, and chloramphenicol antibiotics. This strain was used as an indicator to isolate the phage.
Bacteriophage isolation and plaque morphology
A lytic bacteriophage, designated ɸEcM-vB1 according to a guide for naming and classifying the isolated phage 38, was isolated from sewage water samples against MDR E. coli using spot method and double agar overlay technique. Our results showed that clear plaques appeared at 37°C after 18 h of incubation (Figure 2). After isolation, one plaque was selected for further purification, amplification, and characterization.
Phage host range determination
The host range of ɸEcM-vB1 was evaluated against 65 MDR E. coli isolates and other bacteria including 5 K. pneumonia, 4 A. bauminni and 2 P. aeruginosa. ɸEcM-vB1 phage was capable of infecting 51% (33/65) of the tested E. coli clinical isolates appeared as clear zone. However, isolated phage had a minimal effect on other types of bacteria as shown in Table 2. None of the K. pneumonia and P. aeruginosa isolates were susceptible to our phage. One of the A. bauminni isolates was susceptible to our phage.
Table 2. Host range of ɸEcM-vB1 bacteriophage
Host
|
Susceptibility to ɸEcM-vB1
|
|
Host
|
Susceptibility to ɸEcM-vB1
|
E. coli 1
|
-
|
|
E. coli 39
|
-
|
E. coli 2
|
+
|
|
E. coli 40
|
+
|
E. coli 3*
|
+
|
|
E. coli 41
|
+
|
E. coli 4
|
+
|
|
E. coli 42
|
+
|
E. coli 5
|
+
|
|
E. coli 43
|
+
|
E. coli 6
|
-
|
|
E. coli 44
|
+
|
E. coli 7
|
-
|
|
E. coli 45
|
+
|
E. coli 8
|
-
|
|
E. coli 46
|
+
|
E. coli 9
|
+
|
|
E. coli 47
|
-
|
E. coli 10
|
+
|
|
E. coli 48
|
+
|
E. coli 11
|
-
|
|
E. coli 49
|
-
|
E. coli 12
|
+
|
|
E. coli 50
|
+
|
E. coli 13
|
-
|
|
E. coli 51
|
-
|
E. coli 14
|
+
|
|
E. coli 52
|
+
|
E. coli 15
|
+
|
|
E. coli 53
|
+
|
E. coli 16
|
-
|
|
E. coli 54
|
+
|
E. coli 17
|
+
|
|
E. coli 55
|
+
|
E. coli 18
|
-
|
|
E. coli 56
|
-
|
E. coli 19
|
-
|
|
E. coli 57
|
-
|
E. coli 20
|
+
|
|
E. coli 58
|
-
|
E. coli 21
|
-
|
|
E. coli 59
|
+
|
E. coli 22
|
+
|
|
E. coli 60
|
-
|
E. coli 23
|
-
|
|
E. coli 61
|
+
|
E. coli 24
|
-
|
|
E. coli 62
|
+
|
E. coli 25
|
+
|
|
E. coli 63
|
-
|
E. coli 26
|
-
|
|
E. coli 64
|
+
|
E. coli 27
|
-
|
|
E. coli 65
|
+
|
E. coli 28
|
-
|
|
K. pneumonia 1
|
-
|
E. coli 29
|
-
|
|
K. pneumonia 2
|
-
|
E. coli 30
|
-
|
|
K. pneumonia 3
|
-
|
E. coli 31
|
-
|
|
K. pneumonia 4
|
-
|
E. coli 32
|
-
|
|
K. pneumonia 5
|
-
|
E. coli 33
|
+
|
|
Acinetobacter 1
|
-
|
E. coli 34
|
-
|
|
Acinetobacter 2
|
+
|
E. coli 35
|
-
|
|
Acinetobacter 3
|
-
|
E. coli 36
|
-
|
|
Acinetobacter 4
|
-
|
E. coli 37
|
+
|
|
Pseudomonas 1
|
-
|
E. coli 38
|
-
|
|
Pseudomonas 2
|
-
|
+: indicates that the strain is susceptible to the phage and that clear plaques were produced.
-: indicates that no plaques were observed.
E. coli 3* is the indicator host strain.
Phage morphology
The isolated phage was visualized by TEM (Figure 3). An icosahedral head measuring 63.06 nm in diameter and a long, contractile tail measuring 109.34 nm in length were the features of ɸEcM-vB1 phage. TEM classified the phage as a member of the caudovirales order and the Myoviridae family.
Single-step growth curve analysis
One step growth curve was employed to identify the various stages of the phage infection process, such as the latent period and burst size. Our results indicated that the latent period of ɸEcM-vB1 phage was 10 min, and the average burst size was 271.72 (Figure 4).
Bacteriophage Thermal and pH stability
The stability of ɸEcM-vB1 at various temperatures was investigated, as shown in Figure 5. The results indicated that the virulent phage titer was stable at approximately 5.7 Log10 pfu/ml for 40 min at 40°C. At 50°C, the phage titer decreased after 20 min to 4.85 Log10 pfu/ml. The titer decreased to 3.6 Log10 pfu/ml after 40 min at 60°C, then the phage lost its infectivity after 40 min at 70°C.
The effect of different pH values on phage survival and infectivity was detected using plaque assay, as shown in Figure 6. The phage was able to survive over a broad pH range (3-11), with peak activity at pH 7, where the titer was 7.5 Log10 pfu/ml. Our study revealed greater phage stability at alkaline pH (phage titer is 7.2 Log10 pfu/ml at pH 8) compared to acidic pH (phage titer is 6.8 Log10 pfu/ml at pH 6). Relatively low titers of our phage, with 4.6 Log10 pfu/ml and 4.3 log10 pfu/ml, were observed at pH 3 and 11, respectively. The activity of the ɸEcM-vB1 phage decreased at pH 2 and 12.
Restriction map and protein profile of the isolated bacteriophage
The genome of ɸEcM-vB1, as visualized on 1% agarose gel, was double-stranded DNA of size more than 25 kb as shown in Figure 7A. Our results showed that the genomic DNA was digested by EcoRI (Figure 7B). The isolated phage genome was sensitive only to EcoRI according to enzyme digestion analysis, which also yielded two digest pattern fragments. HindIII and BfaI had no effect on the phage genome producing no fragments. The results of our phage protein profile analysis via SDS‒PAGE are shown in Figure 7C according to a 10 to 250 kDa protein marker. It was estimated that the phage ɸEcM-vB1 had 10 structural proteins with sizes ranging from 22 to 150 kDa.