Polluted and effluent waters typically have an increased diversity of bacteriophages. From fish and shrimp farm effluent water, many bacteriophages infecting Vibrio sp. were discovered [35, 36]. Escherichia phage CMSTMSU, which only infects E. coli with a wide host range, was identified in shrimp farm wastewater in the current investigation. There are several different E. coli that Escherichia phage myPSH1131 can infect, including enterohemorrhagic E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, and uropathogenic E. coli [37]. The clinical isolate of E. coli ATCC 25922 had been successfully infected by the bacteriophage VB EcoS-Golestan [38]. Escherichia phage 590B isolated from Chandigarh, India, sewage water showed lytic activity against an isolate of highly drug-resistant uropathogenic E. coli [39]. Bacteriophages can only infect and multiply in a specific host; they cannot infect or reproduce in all host bacteria. According to Stenholm et al. [40], the host range of phages provides a sensitive method of identifying different bacterial isolates since they primarily replicate in hosts of the same genus [19]. In the present investigation, Escherichia phage CMSTMSU only infects and multiplies in E. coli when it is tested against a variety of hosts, including B. cereus, P. aeruginosa, V. harveyi, V. vulnificus, V. parahaemolyticus, and V. alginolyticus. It might be caused by the interaction between the phages and the host bacteria's cell wall components. The receptor binding protein (RBP) of bacteriophages may be used to identify host surface elements [41]. The outer membrane lipid polysaccharide (LPS) of Gram-negative bacteria is a well-known RBP receptor [42]. Additionally, several coliphages, including OmpC, can identify multiple surface receptors [43].
According to Costerton et al. [44], biofilms are common in aqueous environments and can quickly infect aquatic species and their substrates. However, it is challenging to stop the growth of biofilms because of the structure's resistance to antibiotics and other disinfectants. Other workers have already mentioned biofilm disruption. E. coli biofilm in a glucose-restricted medium was shown to be impacted by bacteriophage T4 by Corbian et al. [45]. Following treatment with bacteriophage obtained from shrimp nursery fluids, Karunasagar et al. [46] were able to reduce the V. harveyi biofilm that had developed on the surface of HDPE. Increased phage concentration gradually reduced biofilm development, whereas Escherichia Phage CMSTMSU successfully inhibited E. coli's ability to generate biofilms. P. aeruginosa bacteriophages were discovered to be capable of wiping off bacteria in a biofilm, according to research by Hanlon et al. [47]. Phages' ability to inhibit biofilm has a number of benefits over antibiotics, including specificity, lack of side effects, anti-toxicity toward microorganisms, etc. The enzyme from the phage that breaks down bacterial polysaccharides may be the cause of the inhibitory mechanism. Polysaccharides are crucial elements in the production of biofilms. According to Doolittle et al. [48], polysaccharide lyases produced by bacteriophages remove polysaccharides from biofilm. It was investigated as a model for managing biofilms. The results obtained showed unmistakably that phage filtrate at higher concentration volumes was very successful in reducing the bacterial populations in the biofilm-formed sheets. The inhibition of bacterial growth on the HDPE surface, which is obviously dependent upon the dosage of bacteriophage, was further strengthened by the unequivocal evidence provided by the confocal laser scanning microscope. The bacteria in biofilms developed antimicrobial substances that shielded the biofilms from phage attack [49, 50]. The phages are found in biofilms because of the usual dietary restriction that occurs in biofilms, which allowed the phage to persist for a long time [51, 49].
In the current investigation, the transmission electron microscope was used to examine the morphology of the isolated Escherichia phage CMSTMSU. The results showed that the isolated phage had an icosahedral head (220–230 nm in diameter) and a non-contractile tail length of 120 nm. According to Rodgers et al. [52], phages with icosahedral heads and lengthy tails might either be members of the Myoviridae or Syphoviridae families. Jurczak-Kurek et al. [19] recovered two phages (MPS1 and MPS2) from sewage and used electron microscopy to analyse their shapes. Because both phages possessed tails, it was previously thought that they belonged to the caudovirales order. Our findings in the current study are consistent with earlier research that revealed the majority of isolated aquatic bacteriophages belonged to the siphoviridae, myoviridae, and podoviridae families [53–55]. The isolated bacteriophage structure in our findings is similar to that in prior investigations. Our isolated phage contains a lengthy, non-contractile tail and an icosahedral head. It has additionally strengthened the strong evidence that it is a member of the siphoviridae family, which was previously validated by numerous other researchers working in other fields of phage study.
The findings of the killing efficiency tests revealed that after 1.30 hours, Escherichia phage CMSTMSU considerably reduced the number of E. coli cells. It might be as a result of the phages' high lytic activity. Our earlier research by Lelin et al. [56] confirmed that the lytic activity of the Vibrio-specific pages effectively reduced the density of V. harveyi cells. After 1 hour and the following 8 hours, the myoviridae vB EcoM-ECP26 phage efficiently reduced the growth of the E. coli NCCP 13930 host bacterial strain [57]. Staphylococcus phages were shown to be absorbed by strains of Bacillus subtilis and Enterococci sp. by Rakieten and Rakieten [58] and Rakieten and Tiffany [59] without appearing to have any impact on the growth of these microorganisms. The adsorption of the phage is followed after a pause by the lysis of the bacterium and a multifold increase in the number of phage particles, which may be the unique interaction between the phage and the host bacteria that causes the inhibition. Additionally, bacteriophages enslave their host's machinery and block its metabolic routes [60]. Upon infecting its host, E. coli, the virulent phage T7 develops approximately 100 offspring phages per host in less than 25 minutes [61].
In the current study, the investigated phages were responsive to a wide range of pH values as well as a variety of temperature ranges. It was thought that pH and temperature affected phage growth. Our Escherichia phage CMSTMSU is capable of withstanding temperatures as high as 50°C. According to Olson et al. [62], temperature has a major impact on bacteriophage survival and is essential for adhesion to host organisms' surfaces, penetration, multiplication, and latent period length. In a similar vein, Xu et al. [63] discovered that phage vB EcoS-B2 was active between 4 and 50°C, but that at 55°C, phage biological activity had drastically decreased. In his work, another phage (SSL-2009a) did not lose activity even at temperatures exceeding 60°C. Escherichia phage vB EcoM-RPN242 isolated from a piglet with diarrhea was capable of remaining stable over a broad temperature range (4–70°C) [64]. Phages' varied functions, which also affect viability, proliferation, and storage conditions, are greatly influenced by temperature [65]. The current findings showed that the Escherichia phage CMSTMSU survived in water from a shrimp farm at temperatures between 40 and 70°C, indicating that the phage had a wide range of tolerance. The V. harveyi siphoviridae-like phage (VHS1), which was discovered in a Thai pond used for raising black tiger shrimp, could withstand temperatures as high as 60°C for two hours and a wide pH range [66]. Escherichia phage CMSTMSU is sensitive to pH, just like it is to temperature. Low pH (4) and high pH (10) have less of an impact on survival than the optimal survival range of 7 to 9. This means that a pH of 7 to 9 may be appropriate for a prospective phage infection. Phage SFP10 was entirely inactive below pH 2, according to Park et al. [67], while it was extremely stable between pH 4 and 10. Phage T7 was reported to be physically stable between pH 6 and 8 by Kerby et al. [68]. When Sharp et al. [69] looked into the pH stability of the T2 phage, they discovered that it was stable over a wide pH range of 5–9. Shiga toxin-producing E. coli (STEC) from food had previously tolerated pH levels between 4 and 10 [57].
The burst size increased in a positive correlation with the rise in burst times, starting at 2% after 20 minutes and reaching a maximum of 115% in 140 minutes. Multiple factors, including the host bacteria's metabolism, the environment, and other factors, affect burst size. Three E. coli bacteriophages—APCEc01, APCEc02, and APCEc03—with latent periods of 60 minutes, 40 minutes, and 40 minutes, respectively, and burst sizes of 90, 30 and 47 phage particles each were the subject of a study by Dalmasso et al. [14]. As crucial elements of the phage infection process, the latent durations and burst sizes of phages were determined using single-step growth studies. The bacteriophage Escherichia phage CMSTMSU has a relatively similar latency duration (about 40 minutes) to other phages [70, 33], but their burst sizes vary greatly (68 pfu). Escherichia phage CMSTMSU's latent and burst periods are different from those of other phages like SSL-2009a, which had latent and burst times of 10–15 and 30–40 minutes, respectively [71]. Even though the latent time of the Escherichia phage CMSTMSU (approximately 10–15 minutes) was somewhat longer than that of SSL-2009a [71], it is still a long latency period. A phage may be regarded as an effective antibacterial agent if it has a large burst size and a brief latent period. It multiplies quickly and could be particularly successful at controlling bacterial diseases because of how quickly it grows [72]. Because of its enormous burst size and brief latent period, the Escherichia phage CMSTMSU is a potent bio-control agent and can be applied in phage therapy.