Bacteriophages: A Possible Solution to Combat Enteropathogenic Escherichia Coli ( EPEC) Infections in Neonatal goats

Kanika Bhargava Amity Institute of Biotechnology, Amity University Rajasthan K Gururaj Central Institute for Research on Goats G. K. Aseri Amity Institute of Microbial Technology, Amity University Rajasthan Gopal Nath Banaras Hindu University Virendra Bahadur Yadav Banaras Hindu University Institute of Medical Sciences R. V. S. Pawaiya Central Institute for Research on Goats Ashok Kumar Central Institute for Research on Goats A.K. Mishra Central Institute for Research on Goats Neelam Jain (  njain1@jpr.amity.edu ) Amity Institute of Biotechnology, Amity University Rajasthan https://orcid.org/0000-0003-1471-7419


Introduction
Escherichia coli are known to be a natural commensal of warm-blooded animal gut ora. A new and more virulent generation of the MDR/XDR/PDR bacteria strain has been developed due to the indiscriminate use of antibacterial agents (Magiorakos et al. 2012). The acquisition of plasmid-borne resistant genes and unregulated antibiotic application has contributed to the emergence of resistance among the Escherichia coli strains. As a highly varied and adaptable pathogen, this peculiar feature of Escherichia coli has resulted and validated from its property of gene gain and loss (Croxen et al. 2013). The pathogenicity of Escherichia coli depends on its virulence factors, invasins, heat-labile and heat-stable enterotoxins, verotoxins and adhesin or colonisation factors (Relman and Falkow 2015). It is pathogenically classi ed into, diarrhoeagenic Escherichia coli and uropathogenic Escherichia coli. Diarrhoeagenic Escherichia coli is further subdivided into Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic Escherichia coli (EPEC), Enteroinvasive Escherichia coli (EIEC), Enteroaggregative Escherichia coli (EAggEC) and Enterohemorrhagic Escherichia coli (EHEC), which causes diarrhoea and other associated illnesses (Garrine et al. 2020). Escherichia coli are present in both humans and livestock, where ruminants (sheep and goats) have been identi ed as a substantial pool and human infection source (Rahman et al. 2020). Since these ruminants not only harbour pathogenic strains, they can also cause asymptomatic infection in animals that can easily pass through the food web and contribute to human clinical diseases (zoonotic diseases). Serotypes like O91, O157 and O146 of EHEC isolated from sheep (Urdhal et al. 2003) and O157 isolated from goats is a liated with human infections (Pritchard et al. 2000). Pathogenic variants of Escherichia coli affect humans and animals, causing diseases worldwide (Croxen et al. 2013), where EPEC is primarily responsible for fatal diarrhoea in developing countries (Bugarel et al. 2011 India ranks second in goat population with nearly 148.9 million approximately (NDDB 2019). In India, goats are considered poor people's bank or insurance scheme, distributed between landless and poor farmers, but the rapid surge in the animal farming sector has led to increased commercial goat farming (Kumar et al. 2010). Goat rearing in India has enormous demand as they are the main meat (chevon) producing animals. Goat farming is gaining traction in the commercial and domestic sectors over the last few years due to its popularity. However, as goat meat and milk are generally recognised worldwide, constraints on the growth of goat enterprises have also been observed due to its association with bacterial (Miller and Lu 2019) and parasitic infections (Ayaz et al. 2018) that are critical for causing human infections (Monteiro et al. 2018) and may pose a threat to public health by spreading zoonotic diseases. A retrospective study on goat from 1988 to 2012 showed that 43.67% of deaths were due to enteritis, followed by gastrointestinal parasitism and gastric diseases (Pawaiya et al. 2017). Medical science is currently entirely dependent on antibiotics to treat infectious diseases and strives to keep the human race healthy and protect animals.
Increase in drug-resistant bacteria has contributed to signi cant morbidity and mortality due to the inappropriate use of antibiotics (WHO 2020). Such conditions have contributed to the use of bacteriophages. The utilisation of broad-spectrum drugs affects the gut microbial community and its composition (Langdon et al. 2016). Structural modi cations contribute to changes in the expression of genes, the activity of the protein, and the gut's overall metabolism, the results of which may or may not be temporary (Hasan and Yang 2019). Bacteria whose genes are altered by broad-spectrum antibiotics may serve as a gene pool for such resistant bacteria, contributing to the spread of antibiotic resistance (Peterson and Kaur 2018). This has prompted researchers to rethink a decades-old phage therapy approach as a viable new therapeutic alternative against bacterial infections. A randomised, double-blind, placebo-controlled clinical trial was performed where bacteriophage cocktails were administered intravesically to treat urinary tract infections (Leitner et al. 2020). Titze et al. (2020) conducted a study where antimicrobial activity was exhibited by phage mixture against Staphylococcus aureus from bovine mastitis. Since bacteriophages are highly selective, they are ineffective against the host non-pathogenic ora (Principi et al. 2019). Phages may be one of those multi-strategic instruments that could modulate microbial diversity and assist us in combating multi-drug resistant bacterial strains as we are about to reach the post-antibiotic period, where we are lagging in the ght against many diseases due to our same old classical approach (Aslam et al. 2018).
Here in this article, we report the isolation of highly pathogenic strains of Escherichia coli, characterised by molecular methods, vis-à-vis their AMR characteristics that could pose a potential risk. Hence, suitable coliphages and their bactericidal activity on indicator organisms were screened to develop suitable phage therapeutic candidates.

Materials And Methods
Sample collection and isolation of E. coli strains from goat kids A study was conducted in hebdomadic goat kids that showed acute diarrhoea with pasty greenish faecal matter soiling the perianal region. Faecal swabs were collected from 32 goat kids born between September-November (kidding season) from various unorganised herds in an organised goat farm. For bacteriological culture, swabs were washed with 1.0ml of sterile PBS and vortexed before inoculation to culture media. They were cultured in MacConkey's agar (MCA) and Eosin Methylene blue agar (EMB) and incubated at 37ºC. The colonies obtained of Escherichia coli were gram stained, and speci c biochemical tests like Indole test and Triple sugar iron agar tests were conducted. The lactose fermenting colonies of MCA were selectively streaked on Congo-Red dye agar and incubated at 37°C for 72 hours, which resulted in the development of colonies with brick red colour.
Molecular characterisation of E. coli by PCR: DNA extraction was performed using the Nucleopore® DNA Kit (Genetix) implementing the manufacturer's protocol from pure sub-cultured MCA colonies. The DNA is then used to detect EPEC and shiga-toxin-producing (Stx) producing E. coli (STEC) based on the conventional PCR (cPCR) detection using bfpA and stx1 gene, respectively. The cPCR primers for bfpA were same as the SYBR green real-time primers designed in-house in the laboratory, while the stx1 gene was ampli ed using stx1F: 5'CACAATCAGGCGTCGCCAGCGCACTTGCT3' and stx1R: 5'TGTTGCAGGGATCAGTCGTACGGGGATGC3' (Talukdar et al. 2013). The identi cation of E. coli molecularly was made by PCR ampli cation of the universal stress protein A (uspA) gene utilising speciesspeci c primers (F-5'-CCGATACGCTGCCAATCAGT-3' & R-5-ACGCAGACCGTAGGCCAGAT-3') ( Fig: 1). The annealing temperature was kept at 55 °C for 1 min. The ampli ed stx1 gene was sequenced by Sanger's dideoxy method using the BigDye terminator kit where the same sample was subjected to DNA isolation for the screening of EPEC by using bfpA SYBR green real-time PCR. The EPEC colonies with brick red colour in Congo-Red dye agar isolated were also used for molecular screening by bfpA SYBR green real-time PCR. Sequence identity plot was done to compare the nucleotide composition and point mutations in the coding region of the STx1 gene of the aboveisolated strain. Cefepime (30µg), Meropenem (10 µg), Piperacillin-tazobactam (100/10 µg), Nitrofurantoin (300 µg), Colistin (10 µg) were tested for the three of the isolated EPEC strains (1873, 1845, B677). In-vitro presence of Extended-Spectrum Beta-Lactamase (ESBL) was also con rmed according to CLSI guidelines. Phenotypic evidence of ESBL development is con rmed when a difference of ≥5mm is observed between the zone diameters of either cephalosporin (ceftazidime) disc and their respective cephalosporin/clavulanate (ceftazidime-clavulanic acid) disc (Wayne, 2011).

Sample collection and isolation of bacteriophages:
For the isolation of bacteriophages, water samples were collected from different Ghats (Assi Ghat, Tulsi Ghat and Manikarnika Ghat) of river Ganges from Varanasi in a sterile plastic container. A sample from the water specimen was treated with 1% chloroform (v/v) for 10 minutes with continuous vortexing/inversion for bacteriophage isolation. It was later centrifuged for 10 minutes at 10778xg. The supernatant, collected was ooded on the 90mm Mueller-Hinton Agar (MHA) lawn culture (4h old EPEC cultured in log phase). It was incubated overnight at 37ºC. After 24h of incubation, the lawn culture is washed with 4 ml TMG (Tris HCL, Magnesium Sulphate, Gelatin pH 7.4) buffer and treated with 1% chloroform vortexing/inverting for 10 minutes. Soon after the chloroform treatment, it is centrifuged for 10 minutes at 10778xg. The supernatant is transferred carefully to another properly autoclaved 1.5 ml centrifuge tube without disturbing the sedimented lysed bacteria. The whole process of centrifugation repeated four times. Again a 4hr old lawn culture of the bacterial host (EPEC) was prepared. As mentioned earlier, the supernatant collected by the procedure was dropped on the plate and was incubated for 24h at 37ºC. After incubating for 18-24hr, the surface with clear plaques was swabbed with TMG buffer by properly autoclaved swab buds and collected in 1.5 ml centrifuge or eppendorf tubes further processed. The same centrifugation process is repeated, as mentioned above, to pellet the bacterial and cell debris at 10778xg for 10 minutes. The puri ed supernatant was collected and was preserved at 4º C for further use.

Isolation of different bacteriophage strains from cocktails:
Cocktail isolated for all the three strains 1873, 1845, B677 is a mixture of different bacteriophages separated into different strains by soft agar overlay method (Kropinski et al. 2009). The soft agar is kept at a molten state at a temperature of not more than 40-43°C in the water bath. Bacterial and phage suspension is added in it which is further poured on the MHA plates and incubated for 18-24h at 37° C. After incubation, different morphological plaques of different sizes are observed which were later cut out and put into Luria-Bertani (LB) broth and was incubated overnight at 37° C. A total of 5 different plaques were cut out, and after incubation, they were treated with 1% chloroform (v/v) for 10 minutes and were centrifuged at 10778xg for 10 minutes. The supernatant obtained was transferred to another 1.5 ml centrifuge tube, and the process of centrifugation is repeated three more times.

Isolation of bacteriophage DNA:
Isolated phage lysate (10 10 PFU/ml) was transferred in an eppendorf, and DNAse (10mg/ml) was added and incubated for 30min at 37°C for degradation of bacterial DNA. After incubation, SDS (Sodium Dodecyl Sulfate) and proteinase K were introduced and was further incubated at 37°C for 1hr. Later, an equal volume of PCI (25:24:1) (Phenol/Chloroform/Isoamyl alcohol) was added and was centrifuged at 10778xg for 10 minutes. The aqueous phase was collected, and then an equal volume of CI (24:1) (Chloroform/Isoamyl alcohol) was added and centrifuged at 10778xg for 10 minutes. RNAse (10mg/ml) was added to the aqueous phase and was incubated for 30 minutes at 37°C. An equal volume of Isopropanol alcohol was added after incubation, and the solution was kept at room temperature. The suspension was centrifuged, and the pellet was washed with 70% ethanol and then again centrifuged at 10778xg for 10 minutes. Pellet was dried at 37°C for 1-2h, and was dissolved in TE (Tris-Cl-EDTA) and was stored at -20°C. The concentration and quality of isolated DNA were measured using a NanoDrop Bioanalyzer spectrophotometer (Thermo Scienti c) at 260nm. Based on phylogenetic analysis, the STEC CIRG1:2017 strain was present in one of the two major branches with close association with STEC strains including ONT: H34, 4756/98, O6E01767 reference strains belonging to various subclasses. The strain 4756/98 was present in the same clade where STEC/CIRG: 2017 was placed. However, the DEC 10J strain was present in the other branch and SWUN 4124 and FD930 strains.

Characterisation of bacteriophages
A sequence identity plot constructed using BioEdit V. 7.2.5 (Hall 1999) of various E. coli mapped for nucleotide variations is presented in Figure: 7. The most common point mutations observed in some of the strains, including CIRG1: 2017 Stx1, are G→A at the 1511 nucleotide position compared to E. coli strain' N1508 Stx1.' Antibiotic susceptibility testing: The isolated strains were multi-drug resistant (MDR) but non-ESBL producing strains of Enteropathogenic Escherichia coli, where all three (1873, 1845 & B677) were resistant to Amoxy-Clavulanic acid, Nor oxacin and Cefepime. Bacterial strain 1873 and 1845 were resistant to Meropenem, and Nitrofurantoin whereas bacterial strain B677 is sensitive to both; 1873 and B677 are resistant to Ampicillin whereas 1845 is Sensitive to same; 1845 and B677 both are resistant to Gentamicin whereas 1873 is sensitive.

Isolation of bacteriophages and its strains:
After dropping supernatant of water specimen on the 3hr old lawn culture of EPEC strain, ambiguous drops are visible after 24hr of incubation which become further comprehensible where complete clearing of the plate is seen (Figure: 8)

Discussion And Conclusion
The prevalence of high morbidity and mortality among neonatal goats in the developing nations is associated with Enteropathogenic Escherichia coli infections ( , which leads to severe, acute diarrhoea coupled with severe dehydration, extreme acid-base and electrolyte imbalance, and mild diarrhoea mortality without systemic disease, often in less than 12 hours (Gruenberg 2014). The present study explores the prevalence of enteric diseases in neonatal goats and their detection via different molecular techniques. Neonatal enteritis is caused by various etiological agents, with the incidence of mixed infections complementing each other and amplifying the condition due to immunity imbalance ). EPEC is a bacterium with many serotypes of which only some are pathogenic, causing diarrhoea and septicaemia, which results in the death of goat kids if left untreated. EPEC isolate detection serves as an indicator for the presence of virulent E. coli in the herd, detected via bfpA gene-based SYBR green real-time PCR. In general the PCR-based detection methods for diarrheagenic Escherichia coli strains of veterinary importance are more adequate, sensitive and rapid in comparison to the classical phenotypic testing methods. The SYBR Green-based detection assays are widely alleged among copious chemistries available for real-time PCR assays (Tajadini et al. 2014). In general, compared to the conventional diagnostic methods, including isolation and culture techniques, molecular tests are highly sensitive, rapid, and less tedious (Franco-Duarte et al. 2019). Early identi cation, diagnosis, and development of a vaccine for EPEC infections, will not only protect neonatal goats from these infections but would also reduce the economic loss burden of the farmers. One of the most common priority areas recognised by national and international agencies is AMR which is proliferating as a silent pandemic (Sharma et al. 2018   In Vitro Assessment of Antibacterial activity of phages against isolated EPEC strains Overlay technique: Different types and sizes of plaques against EPEC Strain