Isolation of bacteriophages from untreated sewage water against multi-drug resistant E.coli - An initiative to fight against drug resistance

Background: Pathogenic Escherichia coli, common drinking water contaminant, cause a large number of morbidity and mortality worldwide. According to the WHO estimates approximately 63,000 annual deaths are due to E. coli infections. Due to selective pressure on coliforms, resistant microbial strains are produced that threatens modern medicine where common infections could become more deadly. So, there is an urgent need to develop alternative anti-microbial to replace existing antibiotics for treating a broad spectrum of bacterial diseases. This revived the interest of scientists in phages as an alternative therapy. Phage therapy is defined as a therapeutic use of bacteriophages (natural predators of bacteria) for treating bacterial infections. In the present study pure phage strain was isolated from the untreated sewage water sample and subjected to 10 fold dilution following double agar layer assay to determine phage titer against multi-drug resistant E.coli following host range analysis and stability testing at varying temperature and pH. Results: Sewage water contains a vast variety of different sizes bacteriophages with clear to diffused boundaries. The pure plaque isolated after repeated plating showed that it was highly specific against tested E. coli strain and could not lyse strains from other species. The titer was calculated to be 109 PFU/ml that remained unchanged at 4°C, 37°C and 50°C temperature. However, at higher pH range phage viability decreases. Conclusions: In future, it would be expected that the isolated bacteriophages could be characterized and used as a therapeutic potential against multi-drug resistant E.coli that not only attenuate superbug spread but could also replace antibiotics. Beside, isolated phages would be utilized as a bio-component in biosensor development against food borne pathogenic bacteria.


Background
Pathogenic Escherichia coli (E. coli), is a common fecal coliform (facultative aerobic Gram negative rods) bacterium. It is a common drinking water contaminant that causes a large number of morbidity and mortality worldwide. According to the WHO estimates approximately 63,000 annual deaths are due to E. coli infections [1]. Beside gastrointestinal tract infections, E. coli is responsible for 8.9% of sepsis cases, 29% of early onset neonatal sepsis cases and the majority of urinary tract infections [2].
Because these organisms are naturally found in human feces in high concentrations, so when the 3 fecal matter gets disposed off and reaches drainage systems where already overused or misused antibiotics released from clinical aspects and agricultural run-offs prevail create pressure on coliforms and produce resistant microbial strains. The persistence of antibiotic resistant microbes not only creates selective pressure on nearby exposed bacteria but also increases opportunities to transfer the resistance genes to associated susceptible bacteria (via horizontal gene transfer involving plasmids, transposons or integrons) that eventually lead to entry into the human food chain [3][4]. This allows them to survive beyond the inhibition or toxicity thresholds. Thus, focuses our interest to develop such anti-microbial that could fight against such situations.
In the present scenario there is an urgent need to develop alternative anti-microbial to replace antibiotics for treating a broad spectrum of bacterial diseases. Action is needed due to an alarming increase of antibiotic resistance that threatens modern medicine where common infections could become more deadly. WHO releases a list of 12 most dangerous superbugs that pose health hazards, E.coli is among one of them [5]. Though worldwide several researchers are working out to fight against it by creating new drugs and other alternatives yet unable to root out the problem. This revived the interest of scientists in phages as an alternative therapy. Phage therapy is defined as a therapeutic use of bacteriophages (natural predators of bacteria) for treating bacterial infections [6].
Beside, phages are the most "safe" and "green" entities applicable for clinical application. Apart from self replication and ability to kill antibiotic-resistant bacteria they are ubiquitous in nature and have high specificity that leads to minimal disruption to normal flora [7]. Unlike antibiotics, whose concentration decreases with time after dosage, phages continuously replicate and infect the target bacteria [8]. The resistance to phages is not transferrable when compared to antibiotics. Also, the isolation of a new phage is relatively fast and cheap compared to the discovery of a new antibiotic.
Phage therapy or use of bacteriophages as therapeutic agents for eradicating bacterial infections was first introduced by preliminary studies of Twort and D'Herelle in the beginning of the 20th century [9][10]. Keeping this in view, present investigation was carried out to isolate pure phage strain against multi-drug resistant E. coli from untreated sewage water.

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Antimicrobial susceptibility testing: The strain was tested against 11 antibiotics and was found to be resistant to five antibiotics which were generally β-lactam and cephalosporin antibiotics (Table1).
Preliminary screening of bacteriophages: In order to confirm the presence of bacteriophages in the sample, preliminary screening using turbidity reduction and streak plate techniques was done. OD of the sample was recorded as 1.9 with respect to control that was 2.7 at 600nm wavelength. As OD of sample decreases when compared to control suggest that bacteriophages present in the sample lyse the bacteria that result in reduction in the bacterial growth and thus turbidity gets reduced. Next, in streak plate technique when 100µl phage lysate was inoculated over the surface of E.coli bacterial lawn on LB agar plates clear regions over the surface of E.coli bacterial lawn on LB agar plates were observed which suggest that sample contains bacteriophages.
Characteristics of bacteriophages present in the sewage: After 48 hours of incubation, each plate was carefully observed for visible plaques. On each plates non uniform several different plaques were formed that were not clearly discernible from each other (more than 300 plaques) and recorded as TNTC (too numerous to count) (Fig. 1). By drawing 8 grids on some plates approximate number of plaques were counted and found to be in the range of 280-1200 (Fig. 2).
To calculate the phage titer of sample less populated plate with 30 to 300 countable plaques was chosen and the number of phage in a solution was determined by calculating the number of plaque forming units per milliliter of phage (PFU/ml) by applying the following formula: PFU per ml = (Plaques per plate) X (dilution factor)/ (Volume of phage plated in ml) The phage titer of the sample was calculated to be within the range of 102 -107 PFU/ml ( Table 2).
On the basis of morphology, 5 different lytic bacteriophages were isolated against E.coli. Morphology of plaque was recorded in terms of size, edge, and boundaries and was noted as small (<1 mm), medium (=1 mm), and large (>1 mm) with clear or diffused type plaques. From each plate, number of plaques were recorded and counted as described earlier ( Table 3).
Isolation of pure phage and determination of phage titers: In order to isolate a single phage strain one of the plate carrying 11 plaques was subjected to repeated plating and each time 10 fold dilution was carried following 20-200 plaques for the next step. After repeating the process 5 times uniform 5 plaques of round shaped clear regions of < 1mm size get clearly visible. To isolate pure phage strain again one of the plaques was picked from the plate having uniform phages and subjected to enrichment followed by DAL. This process was repeated three times (Fig.3). Lastly, 10 fold dilutions were carried out in order to determine the phage titer of pure phage strain. The phage titer for an isolated single phage strain was found to be 6 X109 PFU/ml (Table 4).
Host range determination of isolated pure plaque: In this study, the host range of the isolated pure phage when tested against 7 different gram positive and gram negative bacterial strains (namely,

Staphylococcus aureus, Streptococcus viridans, Streptococcus mutans, Corynebacterium xerosis,
Bacillus cereus, Proteus vulgaris and Pseudomonas aeruginosa) revealed negative results but for ATCC 25922 E.coli strain showed positive results. This suggested that the phage isolated was highly specific.
Isolated pure plaque stability assessment: The thermal and pH tolerance of pure isolated phage was analyzed at different conditions. The phage when treated at 4°C, 37°C and 50°C showed no significant decrease in phage viability thus, are tolerant to these temperatures. But phage showed high resistance against pH treatment. The phage was stable when treated at pH between 3 and 7, while treating the phage with pH 9, result in decreased plaques per plate and with pH 11 a drastic decrease in plaques occurred (Table 5).

Discussion
Extensive antibiotics use for human and veterinary concern, exhibits resistance against many antimicrobial drugs. Previous studies have reported several MDR E.coli strains that exhibit resistance through several mechanisms such as the production of extended spectrum β-lactamases and carbapenemase. Our study showed that the E. coli isolated from clinical sample was resistant to several β-lactam antibiotics that were in accordance with the several studies reported by authors from different parts of the India [11][12][13][14][15]. These E.coli MDR strains not only causes severe infection in humans but also make it difficult to control by using antibiotics. So, Phage therapy using bacteriophage as biocontrol agents is an effective alternative treatment option against multiple drugresistant pathogens. According to the ICTV classification of bacteriophages include 982 different types 6 in Enterobacteriaceae, among which 265 phages belong to Podoviridae, 297 phages to Siphoviridae and 344 phages to Myoviridae [16]. In the present study, lytic bacteriophage against E.coli was isolated from a sewage water sample and phage titer was determined. Turbidity reduction and streak plate methods were used as preliminary screening methods [17] with subsequent processing by DAL method with slight modifications. The protocol used during the study is so simple and easy that it could be used to reproduce best results for the future use. On the basis of morphology, five different lytic bacteriophages were isolated that were in accordance with the previous studies reported and the titer was calculated to be within the range of 102 -109 PFU/ml. The previous study reported phage titer of 2.15 × 109/ml at a dilution of 10-7 [18]. The pure phage isolated when exposed to different host range only form plaques against ATCC 25922 E. coli strain suggesting highly specific characteristic that was in accordance with several findings reported earlier [19][20]. To determine stability of phage under different conditions percent viability with respect to control was analyzed.

Figure 2
Total number of plaques formed in each plate Pure single phage isolation step-by-step.