The indicator S. aureus and 68 regional mastitogenic Staphylococcus isolates of bovine origin were either obtained from the Type Culture Laboratory of the Division of Biological Standardization, Indian Veterinary Research Institute (IVRI), India, or were identified during the current study. S. aureus bacteriophage SA4 isolated from the sewage water of an IVRI dairy farm per the recommended method [19] was obtained from the Laboratory Repository of the Division of Biological Standardization, IVRI, India.

The propagation of the bacteriophage was done by conventional liquid culture method. The 100 mL of 16 to 18 h old pure broth culture of the indicator bacterium (S. aureus) with 0.1% MgSO₄.7H₂O (HiMedia, India) was inoculated with 1.0 mL of the phage suspension. The phage bacteria mixture was incubated at 37℃ with vigorous intermittent shaking until complete lysis (approximately 3 h). The phage lysate was filtered through 0.22 µ membrane filter (EMD Millipore, USA), and stored aseptically in sterile screw capped bottle at 4℃. Genomic DNA of S. aureus bacteriophage SA4 was isolated using a lambda DNA isolation kit (Qiagen, Germany). The LysSA4 gene from the phage was amplified by PCR using specific primers (a kind gift from Dr. D.M. Donovan, United States Department of Agriculture, USA): 5' ATGGCTAAGACT CAAGCAGAA 3' (sense primer) and 5' ATTTACCCGT GTGCCAAG 3' (antisense primer). The PCR reaction consisting of 50 ng of the template DNA, 20 pmol of each primer, and 17 µL of a PCR master mix (Qiagen, Germany) was brought up to a 25-µL volume with nuclease-free water. PCR was performed with an initial denaturation at 94℃ for 5 min followed by 30 cycles of denaturation at 94℃ for 60 sec, annealing at 51.8℃ for 60 sec, extension at 72℃ for 60 sec, and final extension at 72℃ for 7 min. The PCR products were separated in a 1% agarose gel with a 100-bp marker (Thermo Scientific, USA) to determine the gene size.

The LysSA4 gene PCR product was ligated into a pTZ57R/T-TA cloning vector (Qiagen, Germany) and used to transform Escherichia (E.) coli DH5α (Division of Biochemistry, IVRI, India) using a transformation kit (Thermo Scientific, USA). Recombinant clones were preliminarily identified by blue/white colony screening on Luria Bertani agar (HiMedia, India) after overnight incubation at 37℃. Plasmid isolation was carried out using a Plasmid Miniprep kit (Qiagen, Germany) according to the manufacturer's protocol and clone identity was confirmed by digestion at 37℃ overnight with EcoRI (Thermo Scientific, USA). The confirmed clone was then used for sequencing the inserted gene. The gene was sequenced by Chromous Biotech (India) and the sequence was compared with other published lysin sequences in GenBank (National Center for Biotechnology Information, USA) using the ClustalW subprogram of the MegAlign program of Lasergene software (DNASTAR, USA). A multiple sequence alignment report, sequence similarity/divergence data, and phylogenetic tree were generated. Secondary structure predictions and physiochemical properties of LysSA4 in the form of an algorithm were generated with the Protean program (DNASTAR, USA).

The LysSA4 gene was cloned into a pQE30-UA expression vector (Qiagen, Germany) and the endolysin protein was expressed in E. coli M15 competent cells (Division of Biochemistry, IVRI, India) using standard procedures [30]. Briefly, 100 µL of an overnight culture containing the recombinant clones were transferred into 10 mL of Luria Bertani broth (HiMedia, India) containing ampicillin (100 µg/mL; HiMedia, India) and kanamycin (50 µg/mL; HiMedia, India), and incubated in shaker (10 × g; New Brunswick, USA) at 37℃ for 3 h. A 1-mL aliquot of the culture from this stage was taken and kept separately as untreated culture. The remaining cultures were treated with isopropyl β-D-1-thiogalactopyranoside (IPTG; Thermo Scientific, USA) at a final concentration of 1 mM and incubated at 33℃ up to 8 h. A 2-mL aliquot of the culture was harvested every 2 h and the cells were pelleted at 14,000 × g/4℃ for 10 min in a refrigerated microcentrifuge (Sorvall RC5; Thermo Scientific, USA) for SDS-PAGE analysis. The pellet was suspended in 80 µL of 2× sample loading buffer (Merck, USA), boiled for 7 min at 100℃, and centrifuged at 7,000 × g for 10 min at 4℃. The supernatant (20 µL) was loaded with an medium range molecular weight marker [(Merck, USA) and the proteins were separated by minigel SDS-PAGE (Bio-Rad, USA) with a 12% separating gel (Merck, USA) and 5% stacking gel (Merck, USA)]. The time at which the highest level of LysSA4 expression was observed was noted and used as the standard incubation period to achieve a maximum level of expression.

A 50-mL aliquot of the IPTG-treated culture was centrifuged at 4,000 × g for 10 min at 4℃. The cell pellet was resuspended in 2 mL of lysis buffer (100 mM NaH₂PO₄, 10 mM Tris, and 8 M urea, pH 8.0), and kept at room temperature (RT) for 2 h at 5 × g in a shaker incubator (New Brunswick, USA). The bacterial lysate was centrifuged at 10,000 × g for 20 min at 4℃ to remove the insoluble cellular debris. Supernatant containing the recombinant protein was loaded onto 1 mL of a 50% nickel nitrilotriacetic acid agarose slurry (Qiagen, Germany) and mixed gently by shaking 5 × g for 1 h at RT. Next, the mixture was loaded into a syringe column (HMD, India) and the column was washed with 10 mL of wash buffer (100 mM NaH₂PO₄, 10 mM Tris, and 8 M urea, pH 6.3) to remove the non-specific proteins. Protein bound to the resin was then eluted with elution buffer (100 mM NaH₂PO₄, 10 mM Tris, and 8 M urea, pH 4.5) into four eluates of 0.6 µL each. The purified protein was dialyzed against PBS at 4℃ for 48 h. All four eluates were analyzed by SDS-PAGE along with a medium range protein (Merck, USA) to confirm the presence of the protein.

Lytic activity of the protein against its indicator bacteria and the 68 regional mastitogenic Staphylococcus isolates was measured with a spot inoculation test as previously described [25] using the elutes containing protein over lawn cultures of the bacteria. Briefly, each culture of Staphylococcus was inoculated into sterilized BHI broth (HiMedia, India), and incubated at 37℃. The 16 h old pure broth culture of each isolate was spread plated onto the BHI agar (HiMedia, India) and thereafter, 5 µL of the protein preparation was aseptically placed on the dried surface of agar. After overnight incubation at 37℃, the sensitivity of target organism against the phage was observed by formation of clear circular zone.

The lysin gene from phage SA4 was successfully amplified using LysSA4-specific primers. A single 802-bp PCR product was amplified from the phage SA4 genome (Fig. 1A). The amplified product was cloned into a pTZ57R/T-TA cloning vector (Qiagen, Germany). Restriction digestion of the recombinant clone with EcoRI confirmed the presence of the 802-bp sequence within the recombinant plasmid (Fig. 1B).

The cloned LysSA4 gene of S. aureus bacteriophage SA4 was sequenced. The sequence data was submitted to the GenBank database (National Center for Biotechnology Information, USA) and assigned the accession number HQ330981. Sequence analysis of the recombinant clone revealed the presence of a single 802-bp open reading frame. The LysSA4 sequence was then aligned with reported sequences of endolysin genes from other phages. Analysis showed that LysSA4 had a 94.6~97.0% identity with endolysin genes from A5W, G1, GH15, ISP, K, Phi812, and Sb-1 phages (Fig. 2A). Phylogenetic analysis of the nucleotide sequences demonstrated that the endolysin gene of phage SA4 grouped with that of phage GH15 (Fig. 2B), and both of these sequences formed a cluster that was separate from the other sequences.

The deduced amino acid sequence of the LysSA4 gene encoded a partial protein with a calculated molecular mass of 30 kDa. Amino acid sequence of this endolysin gene was then aligned with those of other reported phages. Amino acid sequence analysis indicated that LysSA4 had a 98.5~98.9% similarity with the sequences of endolysin genes from other phages (Fig. 3A). Phylogenetic analysis of the endolysin gene amino acid sequences confirmed the nucleotide sequence analysis findings although the phage SA4 formed a lineage separate from all other reported phages (Fig. 3B). These results indicated that the endolysin genes are conserved among different phages with minor differences.

The 802-bp lysin gene product was amplified from the phage SA4 genome and cloned into a pQE30-UA expression vector. Restriction digestion with EcoRI confirmed the presence of an 802-bp sequence within the recombinant plasmid (Fig. 1B). Transformed E. coli M15 cells were then treated with IPTG and the expressed recombinant lysin (Fig. 4A) fused to a histidine tag was purified by nickel chelating affinity chromatography. SDS-PAGE electrophoresis of the eluted fractions showed an intense protein band of 31 kDa (Fig. 4B).

In the spot inoculation test, the expressed endolysin completely cleared the indicator bacterium (S. aureus isolate IVRI-139; Fig. 5). Lytic efficacy against approximately 18% of total Staphylococcus isolates tested in the study was observed (Table 1). The recombinant endolysin also showed cross-species lytic efficacy (Table 1).