Abstract
Infections due to antibiotic resistant bacteria are increasing globally and this needs immediate attention. Bacteriophages are considered an effective alternative for the treatment of bacterial infections. The aim of this study was to isolate and characterize the bacteriophages that infect Escherichia coli, Klebsiella pneumoniae, and Enterobacter species. For this, clinical bacterial isolates of the mentioned species were obtained from diagnostic centers located in Chennai, Tamil Nadu, India. The bacteriophages were isolated from sewage water samples collected from Tamil Nadu, India. Phage isolation was performed using enrichment method and agar overlay method was used to confirm the presence of bacteriophages. All the phages were characterized for their life cycle parameters, genome analysis, and in vitro phage cocktail activity. The three bacteriophages exhibited broad host range activity: Escherichia virus myPSH2311 infecting E. coli belonging to six different pathotypes, Klebsiella virus myPSH1235 infecting K. pneumoniae belonging to four different serotypes and Enterobacter virus myPSH1140 infecting four different species of Enterobacter. Morphological observations suggested that the bacteriophages belonged to, Phieco32virus (Escherichia virus myPSH2311), Podoviridae (Klebsiella virus myPSH1235), and Myoviridae (Enterobacter virus myPSH1140). The life cycles (adsorption, latent period, and cell burst) of Escherichia virus myPSH2311, Klebsiella virus myPSH1235 and Enterobacter virus myPSH1140 were found to be 26, 40, and 11 min, respectively. Genomic analysis revealed that Escherichia virus myPSH2311 is closely related to Escherichia phage vB_EcoP_SU10, Klebsiella virus myPSH1235 is closely related to Klebsiella phage vB_KpnP_KpV48 and Enterobacter virus myPSH1140 is closely related to Enterobacter phage PG7 and Enterobacter phage CC31. When phage cocktail was used against multiple bacterial mixtures, there was a reduction in bacterial load from 106 to 103 CFU/mL within 2 h. All the three characterized phages were found to have a broad host range activity and the prepared phage cocktails were effective against mixed bacterial population that are resistant to meropenem and colistin, two last resort antibiotics. Infections caused by drug resistant bacteria will be a serious threat in the future and the use of virulent bacteriophages in therapy may offer an effective solution.
Highlights
Bacteriophages are the viruses of bacteria that live in the same ecological niche, where their host bacteria are present (Rohwer, 2003)
Examination of phage morphology by TEM analysis showed that Escherichia virus myPSH2311 had an icosahedral head of 33 ± 3.0 nm, a non-contractile tail length of 65 ± 2.5 nm and belonged to genus Phieco32virus, Klebsiella virus myPSH1235 had the icosahedral head of 80 ± 4.5 nm and very short non-contractile tail that showed the phage belonged to family Podoviridae and Enterobacter virus myPSH1140 had an elongated head of approximately 80 ± 2.0 nm and long contractile tail of 101 ± 3.5 nm in length indicating that it belonged to the family Myoviridae (Figure 1)
The spot test assay showed that the Escherichia virus myPSH2311 had lytic activity against 73% of the tested E. coli isolates, Klebsiella virus myPSH1235 had activity against 52% K. pneumoniae isolates and Enterobacter virus myPSH1140 showed activity against 15 E. cloacae (n = 15), 3 E. hormaechei (n = 4), 2 E. asburiae (n = 4), and 2 E. aerogenes (n = 3) isolates
Summary
Bacteriophages are the viruses of bacteria that live in the same ecological niche, where their host bacteria are present (Rohwer, 2003). Phages are generally very specific (species-specific and strain-specific) to their bacterial host but some phages are polyvalent, and can infect more than one species or strain of bacteria (Chibani-Chennoufi et al, 2004). Phage therapy largely involves the treatment of bacterial infections using bacteriophages/phages (Levin and Bull, 2004). Phages with broad host range are mostly chosen for therapy, because of their broad spectrum host-range activity against multiple bacteria. Phage therapy can be one of the alternatives for combating antibiotic resistant bacterial infections (Rios et al, 2016)
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