TSP, a virulent Podovirus can control the growth of Staphylococcus aureus till 12 hours

TSP, a virulent Podovirus can control the growth of Staphylococcus aureus till 12 hours | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article TSP, a virulent Podovirus can control the growth of Staphylococcus aureus till 12 hours Rabia Tabassum, Iqbal Ahmed Alvi, Muhammad Asif, Abdul Basit, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-170860/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Methicillin-resistant Staphylococcus aureus (MRSA) is a prevailing nosocomial pathogen that causes a large number of diseases in healthcare and community settings. The MRSA causes infections in different tissues of immunocompromised individuals leading to increased morbidity and mortality. It possess various virulence mechanisms to show resistance against to a lot of beta-lactam antibiotics. To tackle this emerging issue of MRSA, there is an urgent need of antibiotic alternatives and utilizing lytic bacteriophages is one of the best promising therapeutic approach. In the present study, a lytic bacteriophage TSP was isolated from hospital wastewater against MRSA. Its morphology, physiology, host specificity, burst size and lytic spectrum were determined and complete genome sequence was analyzed. TSP phage efficiently inhibit bacterial growth for up to 12 hours. TSP phage showed broad lytic spectrum against clinical isolates of MRSA (78%) and MSSA (37%). It showed stability at varying temperatures (25ºC, 37ºC) and pH (5–9), while its maximum storage stability was observed at 4ºC. It had short latent period (20min) and high burst size (103 PFU/ infected cell). TSP genome sequence and restriction analysis revealed that its genome is linear having 17,987 bp in length with an average GC content of 29.7%. The TSP genome showed 98% similarity to S aureus phages SCH1, SCH11 and vB SauP-436A1. According to comparative genomic analysis and phylogenetic tree analysis, TSP phage can be considered as a member of genus “P68viruses”. The strong lytic activity, broad host range and short latent period along with absence of any lysogenic and toxic genes make TSP a very good candidate for phage therapy against MRSA infections if prove safe during in vivo studies. Virology MRSA lytic Bacteriophage Burst size Lytic spectrum Phage therapy P68viruses Host specificity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Antibiotic resistance is one of the major global issues that limit effective treatments against infections with multiple drug-resistant bacteria (MDRB). Antibiotic resistance arise due to several reasons that include inappropriate and overuse of antibiotics, DNA mutations, importation of drug-resistant genes among bacteria and changes in the defense strategies of microbes [ 1 ]. One of the most important gram positive antimicrobial- resistant pathogen is Staphylococcus aureus that causes a large number of clinical infections including skin and soft tissue, infective endocarditis, bacteremia, device-related and respiratory infections both in nosocomial and community settings [ 2 ]. The main reason for antibiotic resistance in S. aureus is changes in penicillin-binding proteins, the formation of autolysin enzymes and excessive and irrational use of antibiotics in health care settings [ 3 ]. The most common antibiotic-resistant group of S. aureus is methicillin-resistant Staphylococcus aureus (MRSA) which shows resistance to a lot of beta-lactam antibiotics, however these organisms are also getting resistance to aminoglycosides, macrolides, fluoroquinolones, chloramphenicol, and tetracycline as well [ 4 ]. The constraints of effective MRSA treatment options with antibiotics frequently leads to the development of chronic infections, which not only leads to increased morbidity and mortality but also prolong hospital stays and higher health care costs as compared to methicillin-sensitive S. aureus (MSSA) strains [ 5 ]. Methicillin-resistant Staphylococcus aureus (MRSA) has become a challenging pathogen because it poses a serious threat for hospitals and the community. Therefore, it is of great concern to develop new strategies that can supplement or replace the utility of existing antibiotics for treatment of MRSA infections. To overcome the problem of antimicrobial resistance, different alternative strategies can be used which include use of bacteriophages, monoclonal antibodies, probiotics and antimicrobial peptides. Among all alternative approaches, bacteriophage therapy is the best alternative approach that can be used to treat multiple drug-resistant S aureus infections. The properties which make bacteriophage therapy the best replacement option include safety, high specificity, and effective lytic activity against bacterial cells [ 6 ]. Compared to synthesis of new antibiotics, production of bacteriophage is cheaper and faster and they can easily proliferate at infection site with limited or no side effects [ 7 ]. S aureus phages have efficient antimicrobial activity as described in various in vitro and in vivo studies [ 8 ] . The phage SLPW and CSA13 isolated from chicken and fecal sewage of pig farm showed a 90% and 92% lytic spectrum against methicillin resistant S aureus strains. Both phages have a short latent period and high burst size. In addition, CSA13 successfully removed S aureus biofilm [ 9 ] while SLPW showed ability to cure MRSA infection in mice [ 6 ]. Furthermore, phages S24-1 and S13 had been isolated from sewage and showed 100% and 89% lytic spectra against clinical isolates of S. aureus [ 10 ] respectively. The current study describes the detailed characterization of lytic TSP phage against S. aureus , isolated from hospital wastewater including virion architecture, thermal and pH stability and complete genome sequence analysis. Host range of TSP was determined against clinical local isolates of MRSA, MSSA, and other non-aureus Staphylococcus strains. The complete genome of TSP is thoroughly characterized for gene annotation and determining DNA homology. Methods Identification and characterization of bacterial strain Different clinical strains of Staphylococcus aureus were isolated from various clinical samples (skin, blood, abscess, wound, anterior nares, catheters and pus discharge) and identified by standard cultural, morphological and biochemical methods [ 11 ]. Clinical sample was collected from Citi Lab, Lahore Pakistan according to standard method of sample collection. Antibiotic susceptibility pattern was determined by Kirby Bauer’s Disk diffusion method on Muller Hinton agar with commercially available cefoxitin (30ug), clindamycin (10ug), erythromycin (15ug), cefotaxime (30ug), oxacillin (5ug), penicillin (6ug), fusidic acid (10ug), vancomycin (30ug), linezolid (30ug) and tigecycline (15ug). The results of antibiotic susceptibility testing were interpreted according to CLSI criteria [ 12 ]. Sequencing of 16S rRNA gene was carried out from Macrogen, Korea. The bacterial strains were further confirmed by analyzing the 16S rRNA gene sequence through BLAST ( http://blast.ncbi.nlm.nih.gov ) and PCR amplification of mec A gene. Isolation of bacteriophage Biochemically and genotypically confirmed methicillin-resistant Staphylococcus aureus strain MR10 was used as host for isolation of bacteriophage from sewage sample collected from Township wastewater effluent, Lahore, Pakistan according to already reported procedure [ 13 ]. The sewage sample was centrifuged (10,000 rpm, 10 minutes) and supernatant was filtered (0.45µm) Subsequently, 25ml of the filtrate was enriched with an equal amount of 2X tryptone soya broth (TSB) containing 10mM CaCl 2 and 2ml of fresh bacterial culture (4 hours old), incubated overnight at 37ºC with constant shaking ( 160 rpm). After incubation, 1% chloroform was added, flask left un-shaken for half an hour at 37ºC, centrifuged (10,000 rpm, 10 minutes) and supernatant was filtered (0.22µm). The filtrate was assessed for lytic activity by spot test and agar overlay method [ 14 ] for determining plaque morphology [ 15 ]. Determination of TSP host range Bacteriophage host range was determined by standard spot assay and efficiency of plating as described earlier [ 16 ] A collection of 32 MRSA strains, 8 MSSA, 4 S epidermidis strains and some species of gram negative organisms ( E. coli , Klebsiella pneumoniae, Serratia marsecence, Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacter cloacae) were used to measure the host range and EOP of bacteriophage TSP. Determination of in-vitro bacteriolytic activity of TSP Bacteriolytic activity of TSP bacteriophage was determined by an already reported method [ 13 ]. An overnight bacterial culture (3x 10 10 cfu) was added into three TSB broth flasks (50ml). TSP bacteriophage was inoculated at MOI-1 and MOI-10 in two flasks and incubated for 24 hours at 37°C in a shaking incubator at 150 rpm. The third flask having only MR10 was incubated under the same conditions which serves as control. The absorbance (OD 600 ) of control and test cultures were assessed for 24 hours with an interval of 2 hours. This assay was performed in triplicate. Determination of Bacteriophage stability at different temperature and pH Storage stability of TSP phage was determined by incubating phage lysate at different temperatures (4, 25, -20 and -80ºC) for 1 month as described earlier [ 17 ]. Bacteriophage stability at different temperatures (25, 37, 45, 50, and 60°C) and a wide range of pH values (4, 5, 6, 7, 8, 9, and 10) was done according to the previously described procedure [ 16 ]. The survival ability of bacteriophage was determined by the double-layer agar technique. Bacteriophage stability experiments were performed by using phage titer 10 10 pfu/ml. Each assay was performed in triplicate. Determination of adsorption assay and one-step growth curve In order to determine the time taken by the TSP phage for adsorbing to the host surface, an adsorption assay was performed as described earlier with some modification [ 18 ]. Phage adsorption was assayed at MOI of 0.1. Percentages of un-adsorbed phages were determined at every 3-minute interval by taking the ratio of PFU/ml to the initial PFU/ml at 0 min in the supernatant. Bacteriophage adsorption rate constant was determined by mathematical formula K= (2.3/Bt) × log (Po/P) [ 19 ]. In order to determine the different phases of the bacteriophage lytic cycle such as latent period, rise period and burst size, one-step growth curve analysis was performed according to the protocol described previously [ 15 ]. Analysis of TSP Genome Phage DNA was extracted from the filtrate by phage hunting protocol previously described [ 17 ]. The isolated phage DNA was analyzed through agarose and quantified through Nanodrop. Bacteriophage genomic DNA was sequenced using illumine sequencing technique from the University of Minnesota, Genomic Centre (UMGC). Reads were analyzed, trimmed and assembled by applying CLC genomic workbench 10. After completing the phage genome assembly, suitable restriction enzymes () were selected from analysis of draft genome sequence to determine whether the TSP phage genome is circular or linear. The isolated phage DNA was double restricted with NcoI and EcoRI (Thermo scientific) and incubated at 37ºC for 6-8 hours. The restriction pattern was analyzed by running on 0.8% agarose gel electrophoresis. Genome annotation was done by using PHASTER (https://phaster.ca) and online RAST server (https://rast.nmpdr.org/). Open reading frames (ORFs) were identified by using Gene Mark and Gene Glimmer ( http://opal.biology.gatech.edu/GeneMark/ ). All the promised open reading frames (ORFs) were confirmed by using online BLASTp (http://www.ncbi.nlm.nih.gov/BLAST). InterProScan Program and Pfam were used for structural domain prediction and motif searches [ http://www.ebi.ac.uk/interpro/search/sequence-search ]. ARNold was used for the detection of potential rho-independent terminators [ 20 ]. The tRNA Scan-SE software was applied for prediction of putative tRNAs [ 21 , 22 ]. The molecular weight of proteins was determined using ExPASy tool (https://web.expasy.org/compute_pi/). The genomic map was constructed through Snapgene software [http://www.snapgene.com/]. The genome sequence of the methicillin-resistant S. aureus phage TSP had been submitted in GenBank under accession no MW286254. Comparative genomic analysis was done by comparing the whole genome sequence of bacteriophage TSP with other phages of Podoviridae , Siphoviridae and Myoviridae family with BLASTN [ 23 ]. Complete genome sequences of phages showed homology with TSP genome were obtained from NCBI data base ( http://www.ncbi.nlm.nih.gov/genbank/ ). Alignment of sequences and phylogenetic tree were made in ClustalW [ 24 ]. The phylogenetic tree of bacteriophage TSP was formulated utilizing translated amino acid sequences of putative genes that encodes major capsid and DNA polymerase with the maximum likelihood method through MEGA7 [ 25 ]. Results Characterization of MRSA strain MR10 Bacterial strain (MR10: accession no. MT272781) isolated from pus discharge was presumptively identified as S aureus based on microscopic examination, biochemical tests, and 16SrRNA sequence analysis. According to CLSI criteria, the MR10 showed resistance to large number of drugs, however, it was sensitive to vancomycin, linezolid and tigecycline (Supplementary material Table 1). BLAST analysis of its 16s rRNA gene sequence showed 98-99% similarity to S aureus strains. Furthermore, mec A gene (310bp) was successfully amplified from its genome (Publication in process). Phenotypically and genotypically confirmed MRSA strain (MR10) was used for isolation of TSP phage. Morphological characterization of TSP bacteriophage A novel lytic phage TSP was isolated from hospital wastewater against MR10. TSP phage formed tiny clear, round plaques (1mm) in diameter (Fig. 1.) TSP phage showed broad host range against MRSA strains TSP phage showed broad lytic activity against MRSA (24 of 32 strains, 78%) and MSSA (3 of 8 strains, 37%). However, it was unable to lyse the tested S. epidermidis strains and isolates of gram negative organisms ( E. coli , Klebsiella pneumoniae, Serratia marsecence, Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacter cloacae ). The plaque formation ability of TSP phage was observed against 18 isolates of MRSA and 1 isolate of MSSA. The efficiency of plating (EOP) of TSP was grouped into four categories; EOP>0.5 for high production, 0.1<EOP<0.5 for medium production, 0.001<EOP<0.1 for low production, and EOP<0.001 for very low production. The higher EOP values of TSP phage against MR5, MR19 and MR26 suggest that these are more susceptible to phage compared with MR10, while remaining 15/19 isolates have low efficiency of plating as compared to the host strain (Supplementary material Table 2). TSP bacteriophage showed strong bacteriolytic activity till 12 hours post-inoculation TSP phage inhibit the bacterial growth for initial 12 hours at MOI-1 and 10 leading to increased bacterial growth after this time in the phage treated mixture but it was still less than growth in the untreated control (Fig 2). TSP bacteriophage highest stability observed at 37ºC and varying pH (5-9) while maximum storage stability at 4ºC To assess the stability of bacteriophage TSP for therapeutic use in the future, its thermal, pH and storage stabilities were analyzed. TSP bacteriophage showed highest stability at temperature 25ºC and 37ºC, however at high temperature (45ºC, 50ºC and 60ºC) a progressive decrease in phage titer was observed which destroyed phage activity at temperature above 60ºC (Fig 3A). The TSP stayed highly active at wide pH range (5 to 9), but under extreme pH (below 5 and above 10) conditions, a marked decrease in phage titer was observed (Fig. 3B). Long term storage stabilities showed that TSP phage was more viable at refrigerator temperature (4ºC) as compared to frozen temperatures (-20ºC and -80ºC). However, TSP phage showed better survival at -80ºC (1.95 × 10 10 ) while a significant reduction in phage titer was observed at -20ºC and 25ºC (Fig 3C). TSP bacteriophage revealed short latent period and higher burst size According to phage adsorption assay, almost 99% of phage TSP could adsorb to the host cell surface within 9 min at 25ºC (Figure 5A). Adsorption rate constant of phage calculated within the interval of 3 to 9 minute is 4.3 x 10 -12 pfu/ml/min. One step growth curve analysis showed short latent period of 20 minutes and average burst size of 103 virions per infected cells (Fig 5B). These results indicated that this phage can rapidly infect the host and replicate. The TSP have a linear genome of 18Kb long To further determine whether the genome of TSB is linear or circular, we determined the 1 site cutter in the phage genome through Neb cutter and found that restriction through NcoI & EcoRI produce four fragments of 9.1, 5.7, 3 and 0.1 kb sizes, if the genome is linear, as shown in figure S1. Digestion of TSP phage DNA through NcoI & EcoRI produced restriction pattern like the proposed pattern by Neb cutter, which confirm that TSP phage DNA is linear (Fig. 5). Also, the restriction pattern confirms that the isolated phage DNA is pure with no other DNA contamination. Genome sequence analysis demonstrates lytic nature of TSP phage Whole genome sequencing and annotation showed that TSP phage consists of a double stranded, linear DNA with a genomic length of 17,987 bp and an average GC content of 29.7%. It contains 20 predicted open reading frames (ORFs) and no tRNA gene. According to BLASTn analysis, the complete TSP phage genome sequence showed 98% identity to S aureus lytic phages SCH1 (Accession No. KY000084.1 ), SCH11 (Accession No. KY000085.1 ) and vB SauP-436A1 (Accession No. MN150710.1 ) with 94% query coverage. The detailed genomic characterization of TSP phage is given in Supplementary material Table S3. All ORFs presented an ATG start codon. Among all 20 ORFs, 12 had assigned functions while the remaining 8 ORFs were annotated as hypothetical proteins. Annotation and functional analysis of predicted ORFs revealed four functional groups: structural (major capsid and scaffold protein, major and minor tail protein, tail fibers protein, collar proteins, structural protein) host lysis (endolysin, holin and CHAP domain-containing protein), DNA manipulation (single stranded DNA-binding protein, DNA polymerase) and DNA packaging protein. Structural proteins and lysis protein are present on the plus strand while DNA manipulation, DNA packaging and maximum hypothetical proteins are on negative strand. The TSP phage genome consists of 5 potential rho-independent transcription terminators. There were no virulence gene detected in phage TSP genome. The open reading frame ORF7 (Endolysin) was considered to be involved in lytic activity against peptidoglycan of host bacterium. According to Pfam and InterPro Scan analysis, endolysin has two polypeptide domains, one is catalytic domain at N terminus called cysteine, histidine-dependent amidohydrolases/peptidase (CHAP) (pfam05257) and (IPR007921), and other is cell wall binding domain at C terminus named as SH3_5 (pfam08460) and (IPR003646). TSP phage endolysin is located between the structural proteins similar to phage CSA13 and this is the unique characteristic of P68 like viruses. TSP phage showed genetic similarity with genus P68virus of family Podoviridae The complete genome sequence of TSP phage was assessed for homology for other S aureus phages (Figure 7A). According to BLASTn analysis, TSP phage showed highest similarity (98%) to phage genomes SCH1, SCH11 and vB SauP-436A1 with 94% query coverage. Comparative genomic analysis indicated that TSP genome showed highest homology with the phages of Podoviridae family, while it showed a distant relationship to the members of other families. According to BLASTp search, major capsid protein of TSP phage showed 99.9% identity to vB SauP-436A1, SCH1 and S13 with 100% query coverage, while DNA polymerase of TSP phage showed 98.95% identity to SCH1 sequence with 100% query coverage (Fig 7B and 7C). Discussion Staphylococcus aureus is a multi-drug resistant infectious agent responsible for a number of morbidities such as abscesses, skin infections, endocarditis and toxic shock syndrome [ 26 ]. Routine antibiotic therapy has been failed to treat infections by MRSA and become a major challenge in the cure of chronic infections. Currently, the exploration of new strategies to supplement existing antibiotic therapy has become a serious objective of research. In the current era of antibiotics resistance, phage therapy is the best possible solution. Our study was aimed to identify the novel virulent bacteriophage against MRSA for controlling the infections of MRSA. A number of studies have been reported on isolation of bacteriophages from sewage water as it is the reservoir of multi drug-resistant bacteria [ 27 ]. The TSP showed lytic spectrum of ~78% and can be considered a phage with relatively broad host range against numerous MRSA strains. In literature, broad host range phages already reported such as P68 (84%) [ 28 ], CSA13 (90%) [ 9 ] and SLPW (92%) [ 6 ]. TSP phage possess strong bacteriolytic action that is crucial for phage therapy. The strong reduction in bacterial growth was observed till 12 hours similar to phage SA97 [ 29 ] at MOI-1 and 10. However in comparison to phage CS1 and DW2, which reduced bacterial growth only for 3 hours [ 30 ], TSP possesses longer inhibitory effect. There was no significant difference between ODs of phage treated group at MOI-1 vs 10 ( p value: 0.47). However, lower MOI is preferred because it might generate lower immune response when applied in the living system. Long term stabilities are vital parameter for any phage preparation to be used for phage therapy [ 31 ]. TSP phage showed best survival ability and performance at physiological temperature of 37ºC which suits it application against MRSA infections. It can withstand the raised temperature till 45ºC but became inactivated at 65 ºC. These results were similar to phages SA2 and SLPW where high temperature progressively inactivated their activity [ 32 ]. It exhibit good pH stability at wide range of pH (5-9), and optimum activity at neutral pH. These results are similar to previous reported studies [ 6 , 33 ]. Tailed phages mostly maintained virion structure and stability under wide range of pH (5-9) [ 34 ]. The inactivity of phage below 4 pH indicates that the denaturation of its structural proteins occurs in acidic environment [ 35 ]. These characteristics may be helpful in administration of phages in different environment as therapeutic agent. We found that phage present highest storage stability at refrigerator temperature similar to results previously reported [ 36 ]. Phage TSP fulfills the ideal parameters of phage therapy that includes short latency period and high burst size. Our finding confirms that the newly isolated phage TSP is a lytic phage with higher lytic activity similar to S. aureus lytic phage SLPW and Stau2 [ 6 , 37 ]. Based on genome length, low G+C content and gene organization, TSP phage is similar to that of well-studied S. aureus lytic phages SLPW, VB_SauP_PhiAG01.3, P66, S13, and SCH1 [ 38-40 , 6 ] which were successfully applied for the treatment of S. aureus infections. Genes involved in structure, DNA replication, packaging and lysis showed best match with other Podoviridae phages listed in supplementary Table S3 [ 41 ]. TSP phage also indicated the only characteristics of S. aureus Podoviridae phages that DNA packaging and DNA polymerase genes present on plus strand while all structural genes located on another strand (Table 2) as described earlier [ 42 ]. It possess the DNA polymerase from B type superfamily, which is a unique feature of Picovirinae subfamily. The classical lysis cassette composed of holin-endolysin system was absent in TSP similar to other Podoviruses [ 43 ], as it possess endolysin between genes for viral morphogenesis. [ 44 ]. Due to absence of evolutionary marker, whole genome sequence and protein sequences of major capsid and DNA polymerase were used to infer the evolutionary relationship of TSP phage [ 9 ]. Comparative genomic analysis and phylogenetic tree analysis of TSP phage showed its close relationship to non-classified Rosenblumvirus phages SCH1, SCH111 and vB SauP-436A. TSP taxonomically classified in to Picovirinae subfamily and P68 genus because it possess the hallmarks of this subfamily ( [ 39 ]. The hallmark of Picovirinae sub family include small genome size (16-19kb), low G + C content (27-29%) and predicted number of genes (20-22) [ 45 ]. Podoviridae S. aureus phages belongs to the genus “P68Virus”, an extremely well-conserved group with respect to nucleotide, amino acid homology, morphology, lytic lifestyle and genome size [ 46 ]. In comparison to Myoviridae and Siphoviridae phages, Staphylococcal phages that belong to Podoviridae family lack diversity and show affiliation to Rosenblumvirus genus and subfamily Picovirinae (68-like viruses) [ 47 , 9 ]. Comparative genomic analysis and phylogenetic tree based on major capsid and DNA polymerase revealed that the newly isolated phage TSP is similar to member of genus “P68virus”. So, it has been placed in Picovirinae subfamily in the family Podoviridae. Conclusion In this study, virulent bacteriophage TSP has been isolated and characterized from sewage water against MRSA. TSP phage showed broad host range, short latency period, and higher burst size. It has strong bacteriolytic activity and capable to resist different conditions of pH and temperature. These are crucial parameters of phage candidates for phage therapy. Whole genome sequencing and annotation along with phylogenetic analysis showed that it’s a member of family Podoviridae. Based on morphological, physiological and genomic characteristics, the TSP phage may be a suitable candidate for the eradication of S aureus infections in humans after successful animal and clinical trials. Declarations Conflict of interest The authors declare that they have no conflict of interest. Ethical approval The article does not contain any studies with human participants and animals performed by any of the authors. Funding This research was supported by Higher Education Commission of Pakistan under National Research Project Program number 4501. Availability of data The genome sequence has been submitted to the NCBI GenBank database (accession no. MW286254). Authors’ contributions R.T. carried out all the experiments and wrote the paper, I.A.I and A.B. performed the genome analysis, M.A. helped in all experiments and S. R. supervised all the experiments and manuscript write up. All authors read and approved the final version of manuscript. Acknowledgements We are very thankful to the Higher Education Commission (HEC) of Pakistan for providing funds (HEC-NRPU-4501) to conduct this research. References Shukla I, Tiwari R, Agrawal M (2004) Prevalence of extended spectrum-lactamase producing Klebsiella pneumoniae in a tertiary care hospital. 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AIMS microbiology 5(3):285 Cui Z, Feng T, Gu F, Li Q, Dong K, Zhang Y, Zhu Y, Han L, Qin J, Guo X (2017) Characterization and complete genome of the virulent Myoviridae phage JD007 active against a variety of Staphylococcus aureus isolates from different hospitals in Shanghai, China. Virology Journal 14(1):26 Jamalludeen N, Johnson RP, Friendship R, Kropinski AM, Lingohr EJ, Gyles CL (2007) Isolation and characterization of nine bacteriophages that lyse O149 enterotoxigenic Escherichia coli. Veterinary microbiology 124(1–2):47–57 Jamal M, Hussain T, Das CR, Andleeb S (2015) Characterization of Siphoviridae phage Z and studying its efficacy against multidrug-resistant Klebsiella pneumoniae planktonic cells and biofilm. Journal of medical microbiology 64(4):454–462 Alvi IA, Asif M, Tabassum R, Abbas Z, ur Rehman S (2018) Storage of Bacteriophages at 4 C Leads to no Loss in Their Titer after One Year. Pakistan Journal of Zoology 50 (6) Hsieh S-E, Lo H-H, Chen S-T, Lee M-C, Tseng Y-H (2011) Wide host range and strong lytic activity of Staphylococcus aureus lytic phage Stau2. Appl Environ Microbiol 77(3):756–761 Głowacka-Rutkowska A, Gozdek A, Empel J, Gawor J, Żuchniewicz K, Kozińska A, Dębski J, Gromadka R, Łobocka M (2019) The ability of lytic staphylococcal podovirus vB_SauP_phiAGO1. 3 to coexist in equilibrium with its host facilitates the selection of host mutants of attenuated virulence but does not preclude the phage antistaphylococcal activity in a nematode infection model. Frontiers in microbiology 9:3227 Kraushaar B, Thanh MD, Hammerl JA, Reetz J, Fetsch A, Hertwig S (2013) Isolation and characterization of phages with lytic activity against methicillin-resistant Staphylococcus aureus strains belonging to clonal complex 398. Arch Virol 158(11):2341–2350 Kornienko M, Kuptsov N, Gorodnichev R, Bespiatykh D, Guliaev A, Letarova M, Kulikov E, Veselovsky V, Malakhova M, Letarov A (2020) Contribution of Podoviridae and Myoviridae bacteriophages to the effectiveness of anti-staphylococcal therapeutic cocktails. Scientific reports 10(1):1–11 Kwan T, Liu J, DuBow M, Gros P, Pelletier J (2005) The complete genomes and proteomes of 27 Staphylococcus aureus bacteriophages. Proceedings of the National Academy of Sciences 102 (14):5174–5179 Gutierrez D, Martínez B, Rodríguez A, García P (2010) Isolation and characterization of bacteriophages infecting Staphylococcus epidermidis. Current microbiology 61(6):601–608 Catalao MJ, Gil F, Moniz-Pereira J, Sao-Jose C, Pimentel M (2013) Diversity in bacterial lysis systems: bacteriophages show the way. FEMS MicroBiol Rev 37(4):554–571 Lobocka M, Hejnowicz MS, Gagala U, Weber-Dabrowska B, Wegrzyn G, Dadlez M (2014) The first step to bacteriophage therapy: how to choose the correct phage. Phage therapy: Current research and applications:23–67 Oliveira H, Sampaio M, Melo LD, Dias O, Pope WH, Hatfull GF, Azeredo J (2019) Staphylococci phages display vast genomic diversity and evolutionary relationships. BMC Genomics 20(1):357 Lavigne R, Kropinski A, King A, Lefkowitz E, Adams M, Carstens E (2012) Family Podoviridae. Elsevier Academic Press, San Diego Gozdek A, Głowacka-Rutkowska A, Gawor J, Empel J, Gromadka R, Łobocka MB (2018) Complete genome sequences of two novel Staphylococcus aureus podoviruses of potential therapeutic use, vB_SauP_phiAGO1. 3 and vB_SauP_phiAGO1. 9. Genome announcements 6 (17) Supplementary Files Supplementarymaterial.docx TSP4.fas Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-170860","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":12111759,"identity":"335551ce-ae5f-4ae3-b80b-ee42b97d0461","order_by":0,"name":"Rabia Tabassum","email":"","orcid":"","institution":"University of the Punjab","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rabia","middleName":"","lastName":"Tabassum","suffix":""},{"id":12111760,"identity":"bd2ee791-4e06-4961-a9f6-b011ae7d00ae","order_by":1,"name":"Iqbal Ahmed Alvi","email":"","orcid":"","institution":"Hazara University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Iqbal","middleName":"Ahmed","lastName":"Alvi","suffix":""},{"id":12111761,"identity":"b3d46601-3e79-42c9-bbe9-32880a7ceb6e","order_by":2,"name":"Muhammad Asif","email":"","orcid":"","institution":"University of the Punjab","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Asif","suffix":""},{"id":12111762,"identity":"1ae6627a-bc70-4932-8d76-ffbcb642c83b","order_by":3,"name":"Abdul Basit","email":"","orcid":"","institution":"University of the Punjab","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Abdul","middleName":"","lastName":"Basit","suffix":""},{"id":12111763,"identity":"c585b630-aeed-41d0-93eb-8a5b286892e0","order_by":4,"name":"Shafiq ur Rehman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDACCQaGDwlgFvMBEMnYAGQR0sI4A6KFLYEELRAWjwFxWuRnNz9seFDGEK3bv+bbYx4GG9kNB3gPG+DTYnDnmGFDwjmG3G033m435mFIM95wgC85Aa8WiQTzB4ltIC1nt0nzMBxO3HCAx/gAXofNSP/YANFy5hlQy3/CWhhu5BhCtJzvYQNqOQDWgt9hN3IKgX6RANrCZm44xyDZeOZhvmS83gc6bGPjjzIboC2Hnz14U2En23e897AEXoeBARtQjUQCG9BSIIeZh7AGoBYg5j/ABuURpWUUjIJRMApGEAAAoB9Qvrcfk30AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1265-3442","institution":"Punjabi University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shafiq","middleName":"ur","lastName":"Rehman","suffix":""}],"badges":[],"createdAt":"2021-01-28 08:43:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-170860/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-170860/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":6102508,"identity":"d126b278-be73-46e2-9795-8ad5919f4340","added_by":"auto","created_at":"2021-02-18 19:02:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":762789,"visible":true,"origin":"","legend":"(A) represent the bacteriophage TSP on the lawn of MR10. (B) Plaque morphology of TSP phage ","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-170860/v1/071fa2ec18c70d47a693c504.png"},{"id":6101939,"identity":"83b30a26-6692-4f08-897e-8bb867f1412c","added_by":"auto","created_at":"2021-02-18 18:56:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89593,"visible":true,"origin":"","legend":"Determination of in-vitro bacteriolytic activity of TSP bacteriophage. Phage treated group; Co-culture MRSA strain in logarithmic phase with TSP phage at an MOI-1 and MOI-10, Control group; MRSA culture without phage TSP, OD of control and phage treated groups were measured at 600nm after an interval of 2 hours for up to 24 hours. The results were obtained from three independent experiments and expressed as means of standard deviation.","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-170860/v1/125b971f8ec1c74d71896e3a.jpg"},{"id":6101622,"identity":"8fd5d39c-a0b3-4941-b13c-b1fad37fb9f1","added_by":"auto","created_at":"2021-02-18 18:50:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":168604,"visible":true,"origin":"","legend":"TSP bacteriophage stability assay. (A) Effect of different temperatures (25, 37, 45, 50 and 60ºC) on stability of TSP phage (B) TSP phage was treated at wide pH range (4, 5, 6, 7, 8, 9 and 10) for 1 h (C) Storage stability of TSP phage at different temperatures (4, 25, -20 and -80ºC) showed maximum survival ability at 4ºC. Experiment was performed in thrice and phage titers were expressed in mean ± standard deviation.","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-170860/v1/bf5705afc510445cbd6ecf61.png"},{"id":6101751,"identity":"271c8bf9-dace-48d9-8ea0-e0eeac089a3c","added_by":"auto","created_at":"2021-02-18 18:53:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":174345,"visible":true,"origin":"","legend":"(A) TSP phage adsorption kinetics. (B) One step growth curve analysis of bacteriophage TSP infecting MR10 at 37ºC. Results were obtained from three independent experiments.","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-170860/v1/3ea485b863e0bf7d02700a8a.png"},{"id":6101746,"identity":"14ebcf3b-4a54-482d-a9d8-1cfca24c4bbc","added_by":"auto","created_at":"2021-02-18 18:53:24","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":55107,"visible":true,"origin":"","legend":"Agarose gel analysis of the TSP phage DNA double digested with NcoI and EcoRI. Lane 1: Restricted TSP phage DNA, Lane 2: Un-restricted TSP phage DNA and Lane M: Lambda phage DNA HindII digested marker (Cat#302005 Bioron).","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-170860/v1/99926ab2ed0c5606dc785d39.jpg"},{"id":6101750,"identity":"b4e47ce1-c7d1-4f2c-8005-5e566a0aae1d","added_by":"auto","created_at":"2021-02-18 18:53:24","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":175082,"visible":true,"origin":"","legend":"Linear genome map of TSP phage. The direction of ORFs were depicted via direction of arrows, four functional groups are present in TSP phage genome and genes in each functional group are represented by different colors, structural gene (yellow), regulatory (blue), host lysis (red), DNA packaging (green), while hypothetical ORFs are indicated in purple.","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-170860/v1/c1eb544e68a4ee60759bf857.jpg"},{"id":6101942,"identity":"b39730a0-d2aa-4282-993e-97292de3e727","added_by":"auto","created_at":"2021-02-18 18:56:24","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":307281,"visible":true,"origin":"","legend":"(A) Comparative genomic analysis of S. aureus phages. Whole genome sequences were aligned by Clustal W. Neighbour-joining method was used to construct phylogenetic tree in MEGA 7. The values at the nodes represents the bootstrap support scores as calculated using 1000 replicates. The triangle highlights the novel phage TSP. Phylogenetic analysis of TSP based on amino acid sequence of major capsid (B) and DNA polymerase (C). Phylogenetic trees were constructed with the alignment tool UPGMA having bootstrap value of 2000. Pseudomonas phage ZC08 and Streptococcus phage C1 act as an out group. The scale bar represent 0.5 and 0.1 fixed mutations per amino acid position. The dark dot highlights newly isolated phage TSP. ","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-170860/v1/e0bbdcf5643ca544d4d65844.jpg"},{"id":15670980,"identity":"06f5cfc1-51c0-4ff3-84ed-7b1ecb92c1c4","added_by":"auto","created_at":"2021-11-18 14:03:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1941419,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-170860/v1/699157b2-c457-4e90-9c1d-74e6c9a41efa.pdf"},{"id":6102259,"identity":"72e455a9-56fc-45ef-aa0b-d5541007283c","added_by":"auto","created_at":"2021-02-18 18:59:27","extension":"docx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":144621,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-170860/v1/46cc0d46941ab82d2e9b3ea6.docx"},{"id":6101756,"identity":"777c2082-6885-4759-a22b-1b1a6fed9de6","added_by":"auto","created_at":"2021-02-18 18:53:24","extension":"fas","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":18665,"visible":true,"origin":"","legend":"","description":"","filename":"TSP4.fas","url":"https://assets-eu.researchsquare.com/files/rs-170860/v1/c9dd6135b69ac445364f79d1.fas"}],"financialInterests":"","formattedTitle":"\u003cp\u003eTSP, a virulent Podovirus can control the growth of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e till 12 hours\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntibiotic resistance is one of the major global issues that limit effective treatments against infections with multiple drug-resistant bacteria (MDRB). Antibiotic resistance arise due to several reasons that include inappropriate and overuse of antibiotics, DNA mutations, importation of drug-resistant genes among bacteria and changes in the defense strategies of microbes [\u003ca href=\"#_ENREF_1\"\u003e1\u003c/a\u003e]. One of the most important gram positive antimicrobial- resistant pathogen is \u003cem\u003eStaphylococcus aureus \u003c/em\u003ethat causes a large number of clinical infections including skin and soft tissue, infective endocarditis, bacteremia, device-related and respiratory infections both in nosocomial and community settings [\u003ca href=\"#_ENREF_2\"\u003e2\u003c/a\u003e]. The main reason for antibiotic resistance in \u003cem\u003eS. aureus\u003c/em\u003e is changes in penicillin-binding proteins, the formation of autolysin enzymes and excessive and irrational use of antibiotics in health care settings [\u003ca href=\"#_ENREF_3\"\u003e3\u003c/a\u003e]. The most common antibiotic-resistant group of \u003cem\u003eS. aureus\u003c/em\u003e is methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) which shows resistance to a lot of beta-lactam antibiotics, however these organisms are also getting resistance to aminoglycosides, macrolides, fluoroquinolones, chloramphenicol, and tetracycline as well [\u003ca href=\"#_ENREF_4\"\u003e4\u003c/a\u003e]. The constraints of effective MRSA treatment options with antibiotics frequently leads to the development of chronic infections, which not only leads to increased morbidity and mortality but also prolong hospital stays and higher health care costs as compared to methicillin-sensitive \u003cem\u003eS. aureus\u003c/em\u003e (MSSA) strains [\u003ca href=\"#_ENREF_5\"\u003e5\u003c/a\u003e]. Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) has become a challenging pathogen because it poses a serious threat for hospitals and the community. Therefore, it is of great concern to develop new strategies that can supplement or replace the utility of existing antibiotics for treatment of MRSA infections.\u003c/p\u003e\n\u003cp\u003eTo overcome the problem of antimicrobial resistance, different alternative strategies can be used which include use of bacteriophages, monoclonal antibodies, probiotics and antimicrobial peptides. Among all alternative approaches, bacteriophage therapy is the best alternative approach that can be used to treat multiple drug-resistant \u003cem\u003eS aureus\u003c/em\u003e infections. The properties which make bacteriophage therapy the best replacement option include safety, high specificity, and effective lytic activity against bacterial cells [\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e]. Compared to synthesis of new antibiotics, production of bacteriophage is cheaper and faster and they can easily proliferate at infection site with limited or no side effects [\u003ca href=\"#_ENREF_7\"\u003e7\u003c/a\u003e]. \u003cem\u003eS aureus \u003c/em\u003ephages have efficient antimicrobial activity as described in various \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies \u003cem\u003e[\u003c/em\u003e\u003ca href=\"#_ENREF_8\"\u003e\u003cem\u003e8\u003c/em\u003e\u003c/a\u003e\u003cem\u003e]\u003c/em\u003e. The phage SLPW and CSA13 isolated from chicken and fecal sewage of pig farm showed a 90% and 92% lytic spectrum against methicillin resistant \u003cem\u003eS aureus\u003c/em\u003e strains. Both phages have a short latent period and high burst size. In addition, CSA13 successfully removed \u003cem\u003eS aureus\u003c/em\u003e biofilm [\u003ca href=\"#_ENREF_9\"\u003e9\u003c/a\u003e] while SLPW showed ability to cure MRSA infection in mice [\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e]. Furthermore, phages S24-1 and S13 had been isolated from sewage and showed 100% and 89% lytic spectra against clinical isolates of \u003cem\u003eS. aureus \u003c/em\u003e[\u003ca href=\"#_ENREF_10\"\u003e10\u003c/a\u003e] respectively.\u003c/p\u003e\n\u003cp\u003eThe current study describes the detailed characterization of lytic TSP phage against \u003cem\u003eS. aureus\u003c/em\u003e, isolated from hospital wastewater including virion architecture, thermal and pH stability and complete genome sequence analysis. Host range of TSP was determined against clinical local isolates of MRSA, MSSA, and other non-aureus \u003cem\u003eStaphylococcus\u003c/em\u003e strains. The complete genome of TSP is thoroughly characterized for gene annotation and determining DNA homology.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eIdentification and characterization of bacterial strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent clinical strains of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e were isolated from various clinical samples (skin, blood, abscess, wound, anterior nares, catheters and pus discharge) and identified by standard cultural, morphological and biochemical methods [\u003ca href=\"#_ENREF_11\"\u003e11\u003c/a\u003e]. Clinical sample was collected from Citi Lab, Lahore Pakistan according to standard method of sample collection. Antibiotic susceptibility pattern was determined by Kirby Bauer\u0026rsquo;s Disk diffusion method on Muller Hinton agar with commercially available cefoxitin (30ug), clindamycin (10ug), erythromycin (15ug), cefotaxime (30ug), oxacillin (5ug), penicillin (6ug), fusidic acid (10ug), vancomycin (30ug), linezolid (30ug) and tigecycline (15ug). The results of antibiotic susceptibility testing were interpreted according to CLSI criteria [\u003ca href=\"#_ENREF_12\"\u003e12\u003c/a\u003e]. Sequencing of 16S rRNA gene was carried out from Macrogen, Korea. The bacterial strains were further confirmed by analyzing the 16S rRNA gene sequence through BLAST (\u003ca href=\"http://blast.ncbi.nlm.nih.gov\"\u003ehttp://blast.ncbi.nlm.nih.gov\u003c/a\u003e) and PCR amplification of mec\u003cem\u003eA\u003c/em\u003e gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of bacteriophage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiochemically and genotypically confirmed methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e strain MR10 was used as host for isolation of bacteriophage from sewage sample collected from Township wastewater effluent, Lahore, Pakistan according to already reported procedure [\u003ca href=\"#_ENREF_13\"\u003e13\u003c/a\u003e]. The sewage sample was centrifuged (10,000 rpm, 10 minutes) and supernatant was filtered (0.45\u0026micro;m) Subsequently, 25ml of the filtrate was enriched with an equal amount of 2X tryptone soya broth (TSB) containing 10mM CaCl\u003csub\u003e2\u003c/sub\u003e and 2ml of fresh bacterial culture (4 hours old), incubated overnight at 37\u0026ordm;C with constant shaking ( 160 rpm). After incubation, 1% chloroform was added, flask left un-shaken for half an hour at 37\u0026ordm;C, centrifuged (10,000 rpm, 10 minutes) and supernatant was filtered (0.22\u0026micro;m). The filtrate was assessed for lytic activity by spot test and agar overlay method [\u003ca href=\"#_ENREF_14\"\u003e14\u003c/a\u003e] for determining plaque morphology [\u003ca href=\"#_ENREF_15\"\u003e15\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of TSP host range \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacteriophage host range was determined by standard spot assay and efficiency of plating as described earlier [\u003ca href=\"#_ENREF_16\"\u003e16\u003c/a\u003e] A collection of 32 MRSA strains, 8 MSSA, 4 \u003cem\u003eS epidermidis\u003c/em\u003e strains and some species of gram negative organisms (\u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae, Serratia marsecence, Pseudomonas aeruginosa, Acinetobacter baumannii \u003c/em\u003eand\u003cem\u003e Enterobacter cloacae)\u003c/em\u003e were used to measure the host range and EOP of bacteriophage TSP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of in-vitro bacteriolytic activity of TSP \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacteriolytic activity of TSP bacteriophage was determined by an already reported method [\u003ca href=\"#_ENREF_13\"\u003e13\u003c/a\u003e]. An overnight bacterial culture (3x 10\u003csup\u003e10\u003c/sup\u003e cfu) was added into three TSB broth flasks (50ml). TSP bacteriophage was inoculated at MOI-1 and MOI-10 in two flasks and incubated for 24 hours at 37\u0026deg;C in a shaking incubator at 150 rpm. The third flask having only MR10 was incubated under the same conditions which serves as control. The absorbance (OD\u003csub\u003e600\u003c/sub\u003e) of control and test cultures were assessed for 24 hours with an interval of 2 hours. This assay was performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Bacteriophage stability at different temperature and pH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStorage stability of TSP phage was determined by incubating phage lysate at different temperatures (4, 25, -20 and -80\u0026ordm;C) for 1 month as described earlier [\u003ca href=\"#_ENREF_17\"\u003e17\u003c/a\u003e]. Bacteriophage stability at different temperatures (25, 37, 45, 50, and 60\u0026deg;C) and a wide range of pH values (4, 5, 6, 7, 8, 9, and 10) was done according to the previously described procedure [\u003ca href=\"#_ENREF_16\"\u003e16\u003c/a\u003e]. The survival ability of bacteriophage was determined by the double-layer agar technique. Bacteriophage stability experiments were performed by using phage titer 10\u003csup\u003e10 \u003c/sup\u003epfu/ml. Each assay was performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of adsorption assay and one-step growth curve\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to determine the time taken by the TSP phage for adsorbing to the host surface, an adsorption assay was performed as described earlier with some modification [\u003ca href=\"#_ENREF_18\"\u003e18\u003c/a\u003e]. Phage adsorption was assayed at MOI of 0.1. Percentages of un-adsorbed phages were determined at every 3-minute interval by taking the ratio of PFU/ml to the initial PFU/ml at 0\u0026thinsp;min in the supernatant. Bacteriophage adsorption rate constant was determined by mathematical formula K= (2.3/Bt) \u0026times; log (Po/P) [\u003ca href=\"#_ENREF_19\"\u003e19\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eIn order to determine the different phases of the bacteriophage lytic cycle such as latent period, rise period and burst size, one-step growth curve analysis was performed according to the protocol described previously [\u003ca href=\"#_ENREF_15\"\u003e15\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of TSP Genome \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhage DNA was extracted from the filtrate by phage hunting protocol previously described [\u003ca href=\"#_ENREF_17\"\u003e17\u003c/a\u003e]. The isolated phage DNA was analyzed through agarose and quantified through Nanodrop. Bacteriophage genomic DNA was sequenced using illumine sequencing technique from the University of Minnesota, Genomic Centre (UMGC). Reads were analyzed, trimmed and assembled by applying CLC genomic workbench 10. After completing the phage genome assembly, suitable restriction enzymes () were selected from analysis of draft genome sequence to determine whether the TSP phage genome is circular or linear. The isolated phage DNA was double restricted with NcoI and EcoRI (Thermo scientific) and incubated at 37\u0026ordm;C for 6-8 hours. The restriction pattern was analyzed by running on 0.8% agarose gel electrophoresis. Genome annotation was done by using PHASTER (https://phaster.ca) and online RAST server (https://rast.nmpdr.org/). Open reading frames (ORFs) were identified by using Gene Mark and Gene Glimmer (\u003ca href=\"http://opal.biology.gatech.edu/GeneMark/\"\u003ehttp://opal.biology.gatech.edu/GeneMark/\u003c/a\u003e). All the promised open reading frames (ORFs) were confirmed by using online BLASTp (http://www.ncbi.nlm.nih.gov/BLAST). InterProScan Program and Pfam were used for structural domain prediction and motif searches [\u003ca href=\"http://www.ebi.ac.uk/interpro/search/sequence-search\"\u003ehttp://www.ebi.ac.uk/interpro/search/sequence-search\u003c/a\u003e]. ARNold was used for the detection of potential rho-independent terminators [\u003ca href=\"#_ENREF_20\"\u003e20\u003c/a\u003e]. The tRNA Scan-SE software was applied for prediction of putative tRNAs [\u003ca href=\"#_ENREF_21\"\u003e21\u003c/a\u003e,\u003ca href=\"#_ENREF_22\"\u003e22\u003c/a\u003e]. The molecular weight of proteins was determined using ExPASy tool (https://web.expasy.org/compute_pi/). The genomic map was constructed through Snapgene software [http://www.snapgene.com/]. The genome sequence of the methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e phage TSP had been submitted in GenBank under accession no MW286254. Comparative genomic analysis was done by comparing the whole genome sequence of bacteriophage TSP with other phages of \u003cem\u003ePodoviridae\u003c/em\u003e, \u003cem\u003eSiphoviridae\u003c/em\u003e and \u003cem\u003eMyoviridae\u003c/em\u003e family with BLASTN [\u003ca href=\"#_ENREF_23\"\u003e23\u003c/a\u003e]. Complete genome sequences of phages showed homology with TSP genome were obtained from NCBI data base (\u003ca href=\"http://www.ncbi.nlm.nih.gov/genbank/\"\u003ehttp://www.ncbi.nlm.nih.gov/genbank/\u003c/a\u003e). Alignment of sequences and phylogenetic tree were made in ClustalW [\u003ca href=\"#_ENREF_24\"\u003e24\u003c/a\u003e]. The phylogenetic tree of bacteriophage TSP was formulated utilizing translated amino acid sequences of putative genes that encodes major capsid and DNA polymerase with the maximum likelihood method through MEGA7 [\u003ca href=\"#_ENREF_25\"\u003e25\u003c/a\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCharacterization of MRSA strain MR10 \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial strain (MR10: accession no. MT272781) isolated from pus discharge was presumptively identified as \u003cem\u003eS aureus\u003c/em\u003e based on microscopic examination, biochemical tests, and 16SrRNA sequence analysis. According to CLSI criteria, the MR10 showed resistance to large number of drugs, however, it was sensitive to vancomycin, linezolid and tigecycline (Supplementary material Table 1). BLAST analysis of its 16s rRNA gene sequence showed 98-99% similarity to \u003cem\u003eS aureus\u003c/em\u003e strains. Furthermore, mec\u003cem\u003eA\u003c/em\u003e gene (310bp) was successfully amplified from its genome (Publication in process). Phenotypically and genotypically confirmed MRSA strain (MR10) was used for isolation of TSP phage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological characterization of TSP bacteriophage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA novel lytic phage TSP was isolated from hospital wastewater against MR10. TSP phage formed tiny clear, round plaques (1mm) in diameter (Fig. 1.)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTSP phage showed broad host range against MRSA strains \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTSP phage showed broad lytic activity against MRSA (24 of 32 strains, 78%) and MSSA (3 of 8 strains, 37%). However, it was unable to lyse the tested \u003cem\u003eS. epidermidis \u003c/em\u003estrains and isolates of gram negative organisms (\u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae, Serratia marsecence, Pseudomonas aeruginosa, Acinetobacter baumannii \u003c/em\u003eand\u003cem\u003e Enterobacter cloacae\u003c/em\u003e). The plaque formation ability of TSP phage was observed against 18 isolates of MRSA and 1 isolate of MSSA. The efficiency of plating (EOP) of TSP was grouped into four categories; EOP\u0026gt;0.5 for high production, 0.1\u0026lt;EOP\u0026lt;0.5 for medium production, 0.001\u0026lt;EOP\u0026lt;0.1 for low production, and EOP\u0026lt;0.001 for very low production. The higher EOP values of TSP phage against MR5, MR19 and MR26 suggest that these are more susceptible to phage compared with MR10, while remaining 15/19 isolates have low efficiency of plating as compared to the host strain (Supplementary material Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTSP bacteriophage showed strong bacteriolytic activity till 12 hours post-inoculation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTSP phage inhibit the bacterial growth for initial 12 hours at MOI-1 and 10 leading to increased bacterial growth after this time in the phage treated mixture but it was still less than growth in the untreated control (Fig 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTSP bacteriophage highest stability observed at 37\u0026ordm;C and varying pH (5-9) while maximum storage stability at 4\u0026ordm;C\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the stability of bacteriophage TSP for therapeutic use in the future, its thermal, pH and storage stabilities were analyzed. TSP bacteriophage showed highest stability at temperature 25\u0026ordm;C and 37\u0026ordm;C, however at high temperature (45\u0026ordm;C, 50\u0026ordm;C and 60\u0026ordm;C) a progressive decrease in phage titer was observed which destroyed phage activity at temperature above 60\u0026ordm;C (Fig 3A). The TSP stayed highly active at wide pH range (5 to 9), but under extreme pH (below 5 and above 10) conditions, a marked decrease in phage titer was observed (Fig. 3B). Long term storage stabilities showed that TSP phage was more viable at refrigerator temperature (4\u0026ordm;C) as compared to frozen temperatures (-20\u0026ordm;C and -80\u0026ordm;C). However, TSP phage showed better survival at -80\u0026ordm;C (1.95 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e) while a significant reduction in phage titer was observed at -20\u0026ordm;C and 25\u0026ordm;C (Fig 3C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTSP bacteriophage revealed short latent period and higher burst size\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to phage adsorption assay, almost 99% of phage TSP could adsorb to the host cell surface within 9 min at 25\u0026ordm;C (Figure 5A). Adsorption rate constant of phage calculated within the interval of 3 to 9 minute is 4.3 x 10\u003csup\u003e-12\u003c/sup\u003e pfu/ml/min. One step growth curve analysis showed short latent period of 20 minutes and average burst size of 103 virions per infected cells (Fig 5B). These results indicated that this phage can rapidly infect the host and replicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe TSP have a linear genome of 18Kb long \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further determine whether the genome of TSB is linear or circular, we determined the 1 site cutter in the phage genome through Neb cutter and found that restriction through NcoI \u0026amp; EcoRI produce four fragments of 9.1, 5.7, 3 and 0.1 kb sizes, if the genome is linear, as shown in figure S1. Digestion of TSP phage DNA through NcoI \u0026amp; EcoRI produced restriction pattern like the proposed pattern by Neb cutter, which confirm that TSP phage DNA is linear (Fig. 5). Also, the restriction pattern confirms that the isolated phage DNA is pure with no other DNA contamination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome sequence analysis demonstrates lytic nature of TSP phage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole genome sequencing and annotation showed that TSP phage consists of a double stranded, linear DNA with a genomic length of 17,987 bp and an average GC content of 29.7%. It contains 20 predicted open reading frames (ORFs) and no tRNA gene. According to BLASTn analysis, the complete TSP phage genome sequence showed 98% identity to \u003cem\u003eS aureus\u003c/em\u003e lytic phages SCH1 (Accession No. \u003ca href=\"https://www.ncbi.nlm.nih.gov/nucleotide/KY000084.1?report=genbank\u0026amp;log$=nucltop\u0026amp;blast_rank=3\u0026amp;RID=6BKKE24G014\"\u003eKY000084.1\u003c/a\u003e), SCH11 (Accession No. \u003ca href=\"https://www.ncbi.nlm.nih.gov/nucleotide/KY000085.1?report=genbank\u0026amp;log$=nucltop\u0026amp;blast_rank=2\u0026amp;RID=6BKKE24G014\"\u003eKY000085.1\u003c/a\u003e) and vB SauP-436A1 (Accession No. \u003ca href=\"https://www.ncbi.nlm.nih.gov/nucleotide/MN150710.1?report=genbank\u0026amp;log$=nucltop\u0026amp;blast_rank=1\u0026amp;RID=6BKKE24G014\"\u003eMN150710.1\u003c/a\u003e) with 94% query coverage. The detailed genomic characterization of TSP phage is given in Supplementary material Table S3. All ORFs presented an ATG start codon. Among all 20 ORFs, 12 had assigned functions while the remaining 8 ORFs were annotated as hypothetical proteins. Annotation and functional analysis of predicted ORFs revealed four functional groups: structural (major capsid and scaffold protein, major and minor tail protein, tail fibers protein, collar proteins, structural protein) host lysis (endolysin, holin and CHAP domain-containing protein), DNA manipulation (single stranded DNA-binding protein, DNA polymerase) and DNA packaging protein. Structural proteins and lysis protein are present on the plus strand while DNA manipulation, DNA packaging and maximum hypothetical proteins are on negative strand. The TSP phage genome consists of 5 potential rho-independent transcription terminators. There were no virulence gene detected in phage TSP genome. The open reading frame ORF7 (Endolysin) was considered to be involved in lytic activity against peptidoglycan of host bacterium. According to Pfam and InterPro Scan analysis, endolysin has two polypeptide domains, one is catalytic domain at N terminus called cysteine, histidine-dependent amidohydrolases/peptidase (CHAP) (pfam05257) and (IPR007921), and other is cell wall binding domain at C terminus named as SH3_5 (pfam08460) and (IPR003646). TSP phage endolysin is located between the structural proteins similar to phage CSA13 and this is the unique characteristic of P68 like viruses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTSP phage showed genetic similarity with genus P68virus of family \u003cem\u003ePodoviridae\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete genome sequence of TSP phage was assessed for homology for other \u003cem\u003eS aureus\u003c/em\u003e phages (Figure 7A). According to BLASTn analysis, TSP phage showed highest similarity (98%) to phage genomes SCH1, SCH11 and vB SauP-436A1 with 94% query coverage. Comparative genomic analysis indicated that TSP genome showed highest homology with the phages of \u003cem\u003ePodoviridae \u003c/em\u003efamily, while it showed a distant relationship to the members of other families. According to BLASTp search, major capsid protein of TSP phage showed 99.9% identity to vB SauP-436A1, SCH1 and S13 with 100% query coverage, while DNA polymerase of TSP phage showed 98.95% identity to SCH1 sequence with 100% query coverage (Fig 7B and 7C).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e is a multi-drug resistant infectious agent responsible for a number of morbidities such as abscesses, skin infections, endocarditis and toxic shock syndrome [\u003ca href=\"#_ENREF_26\"\u003e26\u003c/a\u003e]. Routine antibiotic therapy has been failed to treat infections by MRSA and become a major challenge in the cure of chronic infections. Currently, the exploration of new strategies to supplement existing antibiotic therapy has become a serious objective of research. In the current era of antibiotics resistance, phage therapy is the best possible solution. Our study was aimed to identify the novel virulent bacteriophage against MRSA for controlling the infections of MRSA. A number of studies have been reported on isolation of bacteriophages from sewage water as it is the reservoir of multi drug-resistant bacteria [\u003ca href=\"#_ENREF_27\"\u003e27\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eThe TSP showed lytic spectrum of ~78% and can be considered a phage with relatively broad host range against numerous MRSA strains. In literature, broad host range phages already reported such as P68 (84%) [\u003ca href=\"#_ENREF_28\"\u003e28\u003c/a\u003e], CSA13 (90%) [\u003ca href=\"#_ENREF_9\"\u003e9\u003c/a\u003e] and SLPW (92%) [\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e]. TSP phage possess strong bacteriolytic action that is crucial for phage therapy. The strong reduction in bacterial growth was observed till 12 hours similar to phage SA97 [\u003ca href=\"#_ENREF_29\"\u003e29\u003c/a\u003e] at MOI-1 and 10. However in comparison to phage CS1 and DW2, which reduced bacterial growth only for 3 hours [\u003ca href=\"#_ENREF_30\"\u003e30\u003c/a\u003e], TSP possesses longer inhibitory effect. There was no significant difference between ODs of phage treated group at MOI-1 vs 10 (\u003cem\u003ep\u003c/em\u003e value: 0.47). However, lower MOI is preferred because it might generate lower immune response when applied in the living system.\u003c/p\u003e\n\u003cp\u003eLong term stabilities are vital parameter for any phage preparation to be used for phage therapy [\u003ca href=\"#_ENREF_31\"\u003e31\u003c/a\u003e]. TSP phage showed best survival ability and performance at physiological temperature of 37\u0026ordm;C which suits it application against MRSA infections. It can withstand the raised temperature till 45\u0026ordm;C but became inactivated at 65 \u0026ordm;C. These results were similar to phages SA2 and SLPW where high temperature progressively inactivated their activity [\u003ca href=\"#_ENREF_32\"\u003e32\u003c/a\u003e]. It exhibit good pH stability at wide range of pH (5-9), and optimum activity at neutral pH. These results are similar to previous reported studies [\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e,\u003ca href=\"#_ENREF_33\"\u003e33\u003c/a\u003e]. Tailed phages mostly maintained virion structure and stability under wide range of pH (5-9) [\u003ca href=\"#_ENREF_34\"\u003e34\u003c/a\u003e]. The inactivity of phage below 4 pH indicates that the denaturation of its structural proteins occurs in acidic environment [\u003ca href=\"#_ENREF_35\"\u003e35\u003c/a\u003e]. These characteristics may be helpful in administration of phages in different environment as therapeutic agent. We found that phage present highest storage stability at refrigerator temperature similar to results previously reported [\u003ca href=\"#_ENREF_36\"\u003e36\u003c/a\u003e]. Phage TSP fulfills the ideal parameters of phage therapy that includes short latency period and high burst size. Our finding confirms that the newly isolated phage TSP is a lytic phage with higher lytic activity similar to \u003cem\u003eS. aureus\u003c/em\u003e lytic phage SLPW and Stau2 [\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e,\u003ca href=\"#_ENREF_37\"\u003e37\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eBased on genome length, low G+C content and gene organization, TSP phage is similar to that of well-studied \u003cem\u003eS. aureus\u003c/em\u003e lytic phages SLPW, VB_SauP_PhiAG01.3, P66, S13, and SCH1 [\u003ca href=\"#_ENREF_38\"\u003e38-40\u003c/a\u003e,\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e] which were successfully applied for the treatment of \u003cem\u003eS. aureus \u003c/em\u003einfections. Genes involved in structure, DNA replication, packaging and lysis showed best match with other \u003cem\u003ePodoviridae\u003c/em\u003e phages listed in supplementary Table S3 [\u003ca href=\"#_ENREF_41\"\u003e41\u003c/a\u003e]. TSP phage also indicated the only characteristics of \u003cem\u003eS. aureus\u003c/em\u003e\u003cem\u003ePodoviridae\u003c/em\u003e phages that DNA packaging and DNA polymerase genes present on plus strand while all structural genes located on another strand (Table 2) as described earlier [\u003ca href=\"#_ENREF_42\"\u003e42\u003c/a\u003e]. It possess the DNA polymerase from B type superfamily, which is a unique feature of \u003cem\u003ePicovirinae \u003c/em\u003esubfamily. The classical lysis cassette composed of holin-endolysin system was absent in TSP similar to other \u003cem\u003ePodoviruses \u003c/em\u003e[\u003ca href=\"#_ENREF_43\"\u003e43\u003c/a\u003e], as it possess endolysin between genes for viral morphogenesis. [\u003ca href=\"#_ENREF_44\"\u003e44\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eDue to absence of evolutionary marker, whole genome sequence and protein sequences of major capsid and DNA polymerase were used to infer the evolutionary relationship of TSP phage [\u003ca href=\"#_ENREF_9\"\u003e9\u003c/a\u003e]. Comparative genomic analysis and phylogenetic tree analysis of TSP phage showed its close relationship to non-classified \u003cem\u003eRosenblumvirus\u003c/em\u003e phages SCH1, SCH111 and vB SauP-436A. TSP taxonomically classified in to \u003cem\u003ePicovirinae \u003c/em\u003esubfamily and P68 genus because it possess the hallmarks of this subfamily ( [\u003ca href=\"#_ENREF_39\"\u003e39\u003c/a\u003e]. The hallmark of \u003cem\u003ePicovirinae\u003c/em\u003e sub family include small genome size (16-19kb), low G + C content (27-29%) and predicted number of genes (20-22) [\u003ca href=\"#_ENREF_45\"\u003e45\u003c/a\u003e]. \u003cem\u003ePodoviridae\u003c/em\u003e\u003cem\u003eS. aureus\u003c/em\u003e phages belongs to the genus \u0026ldquo;P68Virus\u0026rdquo;, an extremely well-conserved group with respect to nucleotide, amino acid homology, morphology, lytic lifestyle and genome size [\u003ca href=\"#_ENREF_46\"\u003e46\u003c/a\u003e]. In comparison to \u003cem\u003eMyoviridae\u003c/em\u003e and \u003cem\u003eSiphoviridae \u003c/em\u003ephages, Staphylococcal phages that belong to \u003cem\u003ePodoviridae\u003c/em\u003e family lack diversity and show affiliation to \u003cem\u003eRosenblumvirus\u003c/em\u003e\u0026nbsp;genus and subfamily\u0026nbsp;\u003cem\u003ePicovirinae\u003c/em\u003e\u0026nbsp;(68-like viruses) [\u003ca href=\"#_ENREF_47\"\u003e47\u003c/a\u003e,\u003ca href=\"#_ENREF_9\"\u003e9\u003c/a\u003e]. Comparative genomic analysis and phylogenetic tree based on major capsid and DNA polymerase revealed that the newly isolated phage TSP is similar to member of genus \u0026ldquo;P68virus\u0026rdquo;. So, it has been placed in \u003cem\u003ePicovirinae\u003c/em\u003e subfamily in the family \u003cem\u003ePodoviridae.\u003c/em\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, virulent bacteriophage TSP has been isolated and characterized from sewage water against MRSA. TSP phage showed broad host range, short latency period, and higher burst size. It has strong bacteriolytic activity and capable to resist different conditions of pH and temperature. These are crucial parameters of phage candidates for phage therapy. Whole genome sequencing and annotation along with phylogenetic analysis showed that it\u0026rsquo;s a member of family \u003cem\u003ePodoviridae. \u003c/em\u003eBased on morphological, physiological and genomic characteristics, the TSP phage may be a suitable candidate for the eradication of \u003cem\u003eS aureus\u003c/em\u003e infections in humans after successful animal and clinical trials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe article does not contain any studies with human participants and animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Higher Education Commission of Pakistan under National Research Project Program number 4501.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome sequence has been submitted to the NCBI GenBank database (accession no. MW286254).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.T. carried out all the experiments and wrote the paper, I.A.I and A.B. performed the genome analysis, M.A. helped in all experiments and S. R. supervised all the experiments and manuscript write up. All authors read and approved the final version of manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are very thankful to the Higher Education Commission (HEC) of Pakistan for providing funds (HEC-NRPU-4501) to conduct this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShukla I, Tiwari R, Agrawal M (2004) Prevalence of extended spectrum-lactamase producing Klebsiella pneumoniae in a tertiary care hospital. Ind J Med Microbiol 22(2):87\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKovalskaya NY, Herndon EE, Foster-Frey JA, Donovan DM, Hammond RW (2019) Antimicrobial activity of bacteriophage derived triple fusion protein against Staphylococcus aureus. AIMS microbiology 5(2):158\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHussain MS, Naqvi A, Sharaz M (2019) METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS (MRSA); PREVALENCE AND SUSCEPTIBILITY PATTERN OF (MRSA) ISOLATED FROM PUS IN TERTIARY CARE OF DISTRICT HOSPITAL OF RAHIM YAR KHAN. Professional Medical Journal 26 (1)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFoster TJ (2017) Antibiotic resistance in Staphylococcus aureus. Current status and future prospects. 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Genome announcements 6 (17)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"MRSA, lytic Bacteriophage, Burst size, Lytic spectrum, Phage therapy, P68viruses, Host specificity","lastPublishedDoi":"10.21203/rs.3.rs-170860/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-170860/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMethicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) is a prevailing nosocomial pathogen that causes a large number of diseases in healthcare and community settings. The MRSA causes infections in different tissues of immunocompromised individuals leading to increased morbidity and mortality. It possess various virulence mechanisms to show resistance against to a lot of beta-lactam antibiotics. To tackle this emerging issue of MRSA, there is an urgent need of antibiotic alternatives and utilizing lytic bacteriophages is one of the best promising therapeutic approach. In the present study, a lytic bacteriophage TSP was isolated from hospital wastewater against MRSA. Its morphology, physiology, host specificity, burst size and lytic spectrum were determined and complete genome sequence was analyzed. TSP phage efficiently inhibit bacterial growth for up to 12 hours. TSP phage showed broad lytic spectrum against clinical isolates of MRSA (78%) and MSSA (37%). It showed stability at varying temperatures (25\u0026ordm;C, 37\u0026ordm;C) and pH (5\u0026ndash;9), while its maximum storage stability was observed at 4\u0026ordm;C. It had short latent period (20min) and high burst size (103 PFU/ infected cell). TSP genome sequence and restriction analysis revealed that its genome is linear having 17,987 bp in length with an average GC content of 29.7%. The TSP genome showed 98% similarity to \u003cem\u003eS aureus\u003c/em\u003e phages SCH1, SCH11 and vB SauP-436A1. According to comparative genomic analysis and phylogenetic tree analysis, TSP phage can be considered as a member of genus \u0026ldquo;P68viruses\u0026rdquo;. The strong lytic activity, broad host range and short latent period along with absence of any lysogenic and toxic genes make TSP a very good candidate for phage therapy against MRSA infections if prove safe during \u003cem\u003ein vivo\u003c/em\u003e studies.\u003c/p\u003e","manuscriptTitle":"TSP, a virulent Podovirus can control the growth of Staphylococcus aureus till 12 hours","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-18 18:50:21","doi":"10.21203/rs.3.rs-170860/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b0e1594b-423e-4774-9fe4-f631fec0b11f","owner":[],"postedDate":"February 18th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2478545,"name":"Virology"}],"tags":[],"updatedAt":"2021-09-02T03:29:47+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-18 18:50:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-170860","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-170860","identity":"rs-170860","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}