MATERIALS & METHODS

solation of A. veronii and its antimicrobial sensitivity pattern

Bacterium A. veronii was isolated fr om fish cul- ture pondwater around Hisar (Haryana, India) by surface inoculation of 100 µl of water sample over Starch-ampicillin agar (HiMedia) media and subsequent incubation at 35±1°C for 18-24 h. Yellow to honey-colored colonies of typically 3-5 mm diameter (suspected A eromonas spp.) were also indicative of amylase positive reaction (presence of clear zone) after flooding with 5 ml of Lugol iodine solution 13 . These suspected A eromonas colonies were identified phenotypically and biochemically and further confirmed on the basis of PCR amplified gyrB gene (~1100bp fragment) sequencing. Kirby-Bauer’s procedure for antimicrobial resistance (AMR) assay using disc diffusion and double disc synergy methods was performed on Mueller-Hinton agar (HiMedia) media.

Isolation of lytic bacteriophage

Lytic phage specific to multidrug-resistant A eromonas veronii was isolated fr om enr ichment of same pondwater samples with overnight grown bacterial culture into 5X Nutrient broth (HiMedia) at 37°C for 18-24h. After incubation, the mixture was centrifuged at 10000 x g for 10 min. and supernatant was filtered through sterile 0.22 µm Millex ® -GV syringe filter unit (Merck, Germany). Phage was isolated by spot test with 10µl of filtered sample followed by incubation at 37°C for 16-18 h. Putative A . v eronii phage produced a clear zone in the spotted area.

Phage stock preparation and its titre enumeration

The lytic phage so visible was subjected to purification by selecting a single isolated plaque (~3mm diameter) and suspending it in 0.1 ml of SM buffer (50 mM Tris-Cl, pH 7.5, 99 mM NaCl, 8 mM MgSO4, 0.01% gelatin). Then, serial dilution technique was performed for the bacteriophage suspension followed by double agar layer plaque assay three consecutive times to obtain clonal selection. Further, titre estimation was done as described previously 2 . For phage stock preparation, freshly suspended phage was added to exponentially growing host culture and subsequent overnight incubation was given for 16-18 h. The bacterial lysate was the treated with chloroform, Dnase I and Rnase A in order to cause host inactivation and release of free phages in the suspension. Further, phage was precipitated using Polyethylene glycol and finally chloroform extraction was performed. Preservation of the isolated phage was carried out at -80°C as glycerol stock in National Centre for Veterinary Type Culture (NCVTC) with accession no. VTCCBPA 265.

iological spectrum of phage Aeromonas phage φ265

Different A eromonas spp. strains were subjected to spot test with the newly isolated bacteriophage Different tested species included various A . veronii isolates, following incubation, a clea r lytic zone in the spotted area was considered as indicative of a clear lysis.

Sensitivity assay to chloroform, temperature and pH

Phage Aeromonas phage φ265 was incubated for 1 h at room temperature after treatment with chloroform dilution (5, 10, 20 and 30%) and the % inactivation was accounted as compared to the phage suspension kept in SM buffer held at 4°C. Phage stability assay at different temperatures (4, 25, 37, 45, 55, 60 and 80°C) was examined by incubation of the phage at each temperature for 1 h followed by titre estimation at the end of the incubation. Phage preparation held at 4°C acted as control. For stability evaluation of bacteriophage at various pH values, pH of the solution was adjusted with either 1 M HCl or 1 M NaOH to reach a desired pH range of 3–10. This phage suspen- sion was then incubated at 37°C for 1 h. Phage suspension held at pH 7 acted as control. Double

One step growth curve

The one-step growth experiment was performed as described by Duarte et al., 2018. Briefly, a mid -log phase bacterial culture of A . veronii (OD 600 nm =0.51) (9 ml) grown in Nutrient broth was incubated with 2 ml of phage BPA 265 at an MOI of 0.001 for adsorption without shaking at 37°C for 15 min. Thereafter, the mixture was centrifuged and pellet was resuspended in 10 ml sterile Nutrient broth at time zero (t=0). The tube contents were incubated at 37°C in a shaker incubator at 180 rpm for 2h. 0.1 ml aliquot was drawn at 10 min interval each and subjected to phage titration using double agar plaque assay after se- rial dilution in SM buffer. Plaques were counted after overnight incubation at 37°C.

RESULTS

Antimicrobial resistance pattern of A. veronii

The isolate was found to be resistant to four of the 15 tested antimicrobials according to a standard procedure based on the guidelines of the Clinical and Laboratory Standards Institute (CLSI) and found to be ESBL (Extended spec- trum β-lactamases) and AmpC β-lactamases pos- itive as determined by the double disc synergy methods.

Plaque morphology

A single plaque was isolated and purified against A. veronii. The pla que was having a 1mm lytic centre and the plaque morphology was uniform on all the nutrient agar plates with a marginal or no difference in the diameter (Fig I) and each plaque was characteristically surrounded by a turbid ring (bacterial growth) which was in turn surrounded by a lytic ring (Fig II). The average plaque diameter of phage was ~3mm.

Bacterial host range

The Aeromonas phage φ265 produced clear lytic zone on all of the different A . veronii isolates tested barring one as indicated in Table I.

Phage stability assays

Aeromonas phage φ265 was found to be relatively heat stable over a period of 1 h, between a temperature range of 4-55°C. However, at 60°C, a sharp decrease in phage activity was observed. When exposed to 80°C temperature, after 1 h a complete phage inactivation was observed Moreover, 37°C was found to be the optimum temperature for phage activity. Similarly, pH 7 was found to be the optimum and phage activity decreased while moving to both extremes, however, no complete inactivation was observed (Fig IV). After chloroform treatment, the infectivity of the phage remained similar to its original infectivity, indicating its resistance to chloroform for the tested range.

One step growth curve

As observed from the one step growth curve ex- periment, a triphasic growth curve depicting eclipse, latent and exponential growth period was observed. The latent period of the phage was ~30 minutes and average burst size was 40 PFU infected cell -1