Isolation and characterization of Salmonella paratyphi phage and its lytic spectrum that correlation with pili

Abstract


Abstract
Background Prevention and control of Salmonella bio lm have great importance for economic point and medical. Phages and their derivatives are ideal candidates for replacing or compensating of antibiotic problems in the future.

Results
In this study, the phage KM16 was isolated from slaughterhouse sump samples. It belonged to the Myoviridae family and optimal growth temperature was 42 ℃, the pH of optimal preservation buffer was 6 ~ 7, optimal multiplicity of infection (MOI) was 0.0001 and the genome size was 170,126 bp. The phage KM16 has the ability to lytic most clinical strains of Salmonella paratyphi A and Salmonella paratyphi B. Phylogenetic analysis found that the 16S rRNA, crispr 1 and mA genes of Salmonella paratyphi have a high similarity and correlation with lytic spectrum of phage KM16, but not correlated with the genes of invA, isrK and luxS. Above all, the lytic spectrum of phage KM16 correlation with tertiary structure of Salmonella pili, the pili of Salmonella was the recognition site for phage adsorption. Collaboration of phage KM16 and antibiotics have better anti-bio lm effect than alone of phage or antibiotics in low concentration of bacteria culture and phage have better anti-bio lm effect than antibiotics in a high concentration of bacteria culture.

Conclusions
The data of this study provided a new perspective to understand the relationship between phage lytic spectrum and difference of host strains.

Background
Salmonella as the most serious foodborne pathogens among worldwide and it was distributing widely over natural environment, in addition, it was widely existed on all kinds of food and food raw materials [1]. Salmonella mainly lodges in common domestic animals, and humans are also one of its main hosts. It's a serious zoonosis original microbes, which not only cause disease in livestock, poultry animals, rats and mice, but also cause of food poisoning in human [2]. Majowicz and Deng have reported there are 94 million gastroenteritis cases and 155, 000 people deaths caused by Salmonella in the world every year [3,4].
Bio lm was a multicellular community of microorganisms where microorganisms are embedded selfproduced extracellular matrix and attached to highly hydrated extracellular matrixon, non-biological and biological surfaces [5][6][7]. The extracellular polymeric substances matrix of bio lm acts as a barrier that reduces the penetration of antimicrobial agents and microbiotic into the interior of the bio lm [8,9].
Bio lm of microorganism are highly resistant to desiccation, heat, antibiotics and acidic condition [10].
Bacteria in the bio lm are approximately 10 to 1000 times less sensitive to antimicrobial agents than planktonic bacteria, on account of extracellular polymeric substances of the bio lm that prevent contact with antimicrobial agents [11,12]. This makes totally eliminate of bio lm in clinic, food industry and husbandry are scarcely possible [13].
Penicillin has been around since 1943, since then antibiotics play an important role in controlling bacterial infections and protecting human health. Nevertheless, with the aggravation of antibiotic abuse, widespread drug resistance problems, adverse consequences and serious harm have been caused. As early as 2003, statistics from the ministry of health, PRC showed that the annual death toll caused by the abuse of antibiotics in China was as high as 80,000. The food industry and clinic faces a serious Salmonella contamination problem, what is more exacerbated by the overuse of antibiotics, resulting in an increasing number of antibiotic-resistant foodborne and clinic Salmonella. Efforts to develop new ways to control Salmonella contamination in food and its processing environment are important. The applications of antibiotics and disinfectants at stationary phases to eradicate Salmonella bio lms could be have adverse effects on human health [14].
Phages and their derivatives can be used as a novel, feasible, and safe biological product for the prevention, treatment, and elimination of Salmonella in food, clinic and food processing environments. Phages and their derivatives are ideal candidates for replacing or compensating for antibiotic problems of the future [15]. Phages are viruses that with bacterial lysis activity [16,17]. Due to the ability of kill bacteria, which appear to be a good alternative to antimicrobials and disinfectants [18]. Above all, phages infect only bacteria and not be harmful to humans, making them safe for apply to clinic and food products [19]. Recent study found that that phage has high e ciency in reducing and control bacterial bio lms on various surfaces formed by Escherichia coli, Salmonella, Listeria monocytogenes and Pseudomanas aeruginosa [20][21][22][23][24].
Prevention and control of contamination caused by multidrug-resistance (MDR) bacteria of Salmonella has great importance for an economic point of view and medical [25]. The main challenges for such a phage therapy of the relatively narrow lytic range against bacterial strain [26]. Here we report a new isolated lytic phages named KM16 have better anti-bio lm effect than antibiotics in high concentration of bacteria. In addition, phylogenetic analysis found that the lytic spectrum of phage KM16 correlation with tertiary structure of Salmonella pili, the pili of Salmonella was the recognition site for phage adsorption.

Results
Characteristics and morphology of isolated phages Virulent phage KM16 was isolated from the samples of slaughterhouse sump that according to the host of Salmonella paratyphi NA3. The plaque of phage KM16 was appeared 1 mm in diameter after overnight incubation at 37℃ (Figure 1).
Negatively stained of puri ed phage KM16 was observed with an electron microscope. Transmission electron microscopy (TEM) revealed KM16 virions with an icosahedral head 110 ± 5 nm in diameter, and a non contractile tail 110 ± 5 nm long ( Figure 2). The morphology of phage KM16 indicated they belonged to the Myoviridae family.
After standing for 5 min at 15 ℃, nearly 85% of phage particles were adsorbed to the host bacteria, after incubation for 20 min, almost all phages were adsorbed to host bacteria ( Figure 3). A growth curve of the phage KM16 was obtained by inoculation on Salmonella paratyphi A NA3 according to MOI of 0.1 at 37℃ (Fig. 3). The latent period of phage KM16 was 60 min. The titer of phage KM16 were explosive growth before 170 min and reached peak at 9 h, and then it's appear going down at 15 h later. The ampli cation factor of phage KM16 was approximately 200 times.
Optimum temperature, pH and MOI of isolated phages The phage titer of KM16 was measured by the double plate method at different temperature after 1 h of processed. KM16 have the highest activity after treatment for 1 h at 42℃, then there was a noticeable decline at 50 ℃ and complete inactivation until 90 ℃ ( Figure 4). The result show that phage KM16 have low temperature adaptability and which consistent with the optimum survival temperature of it's host.
Phage KM16 have the most plaque at pH=6~7, further, plaque at pH=10 ~11 were signi cantly higher than that at pH3~4 (Figure 4). These results indicated that the phage KM16 have good tolerance to alkali but extremely intolerant to acids.
Multiplicity of infection (MOI) refers the ratio of the number of phages to cells. The optimum MOI of phage KM16 were 0.0001, among them, the plaque of KM16 were decreased signi cantly after 0.001 and reach minimum at MOI=100 (Fig. 4).

DNA extraction restriction endonuclease digestion and genome analysis
The genome size of phage KM16 is 170,126 bp. We have identi ed 278 protein-coding genes for KM16 genome (Table S1). KM16 genome DNA was digested by EcoRI, Hind III, NotI and Xhol I. This is consistent with the results of phage genome analysis of that phage KM16 has more endonuclease sites. Genome analysis revealed that phage KM16 was virulent phages ( Figure 5,6,).
Phages bacteriostasis spectrum, host resistance and phylogenetic analysis the genes of 16S, host bio lm and crispr

Antimicrobial susceptibility
The Salmonella paratyphi A and Salmonella paratyphi B were isolated from clinical patients in the laboratory of rst people's hospital of Yunnan province. Unfortunately, they have a broad spectrum of resistance (Table 1), but fortunately, most of them can be removed by the phage of KM16 (Table 2). They are all possess resistant to penicillin, streptomycin, cefoxitin, gentamicin, cipro oxacin and tobramycin, but sensitive to kanamycin sulfate and macrodantin.
The lytic phage of Salmonella paratyph KM16 was able to infect most strains of Salmonella paratyphi which were isolated from The First People Hospital of Yunnan Province, China ( Table 2). The Salmonella paratyphi A JJ9 and Salmonella paratyphi A NA3 exhibited sensitivity to KM16. This analysis underlined the wide host range of the isolated phage KM16.
The genes of 16S rRNA (bacterial systematics), crispr 1 (Clustered regularly interspaced short palindromic repeats, an immune weapon produced by bacteria and phages ghting each othe), mA (Salmonella pili), invA (Virulence genes), isrK (Quorum sensing gene) and luxS (Quorum sensing gene) of Salmonella paratyphi were aligned for phylogenetic analysis. Phylogenetic tree analysis indicated that genes of 16S, crispr 1 and mA of Salmonella paratyphi have a high similarity and correlation with lytic spectrum of phage KM16, but not correlated with genes of invA, isrK and luxS. The results indicate that the relatedness, immune system and pili of host were corresponded with the lytic spectrum of phage, nevertheless, the virulence, and quorum sensing of host were not corresponded with the lytic spectrum of phage ( Figure 7).

Structure of Salmonella mA (pili) protein
The pili of Salmonella was the recognition site for phage adsorption. Comparison of Salmonella mA among Salmonella paratyphi A-A, Salmonella paratyphi A-NA3 and Salmonella paratyphi B-H using SWISS-MODEL (https://swissmodel.expasy.org/) for tertiary structure prediction. SWISS-MODEL searches with other Salmonella mA with experimentally solved tertiary structure revealed a signi cant match (https://www.rcsb.org/structure/6erj) ( Figure 8). There were an signi cant differences tertiary structure of Salmonella pili among phage KM16 sensitive and resistant strains ( Figure 8).

Compare the effects of phages and antibiotics on host bio lms
The effects of KM16 (MOI=0.1) and kanamycin sulfate (10 µg/mL) on round coverslip of Salmonella paratyphi A NA3 bio lm were assessed through Scanning electron micrograph (SEM). In this case of Salmonella paratyphi A NA3 seed solution inoculation at a rate of 1/250, added KM16 (MOI=0.1) and kanamycin sulfate (10 µg/mL) in immediately and culture for 24 h, antibiotics have better sterilization effect than phages whether in the results of scanning electron micrograph, OD 600 of bacterial culture solution, or microplate reader OD 570 of bacterial bio lm ( Fig. 9, 10).
Nevertheless, In another case of Salmonella paratyphi A NA3 seed solution inoculation at a rate of 1/250 and culture for 12 h later, then KM16 (MOI=0.1) and kanamycin sulfate (10 µg/mL) were added and cultured for 12 h, phages have better sterilization effect than antibiotics whether in the results of scanning electron micrograph, OD 600 of bacterial culture solution, or microplate reader OD 570 of bacterial bio lm (Fig. 9, 10). In addition, in case of of Salmonella paratyphi A NA3 seed solution inoculation at a rate of 1/250 and culture for 12 h later, then KM16 (MOI=0.1) and kanamycin sulfate (10 µg/mL) were added and cultured for 12 h, collaboration of phage and antibiotics have better sterilization effect than alone of phages or antibiotics whether in the results of scanning electron micrograph, OD 600 of bacterial culture solution, or microplate reader OD 570 of bacterial bio lm (Figure 9,10).
The result of host colony-forming unit indicate collaboration of phage and antibiotics have better sterilization effect than alone of phage or antibiotics ( Figure 11). Meanwhile, under the case of Salmonella paratyphi A NA3 seed solution inoculation at a rate of 1/250 and culture for 12 h later, then KM16 (MOI=0.1) and kanamycin sulfate (10 µg/mL) were added and cultured for 12 h, phages have better sterilization effect than antibiotics ( Figure 11).

Discussion
Salmonella is a common foodborne pathogen, which mainly found in contaminated food and cause severe medical problems and foodborne diseases [27,28]. The Salmonella paratyphi A NA3 used in this study was isolated from a clinical patient in the rst people's hospital of Yunnan province, China who accidentally ate Salmonella contaminated bread. It's regrettable of that overuse of antibiotics has increased the severity of salmonella [29]. The main reasons of drug resistance was that antibiotic resistance (AR) and multidrug-resistance (MDR) genes were in the genome of almost all Salmonella strains [30].
The isolated Salmonella phage KM16 belongs to Myoviridae and the genome sizes was 170, 126 bp. Corresponding, the geonme sizes of Salmonella phage ΦStp1 was 112,149 bp and Salmonella phages SPFM was 233 to 242 Kb, the consistent part was they all belong to the Myoviridae family [31,32].
With the rise of antibiotic abuse, multiple resistant bacteria and superbacteria hence a public health hazard. Alternatives to antibiotics are urgently needed, phage, are dawn of this increasing drug resistance. The isolated Salmonella phages KM16 were ideal substitute for antibiotics for they can lysin multidrug-resistant bacteria. What's more, phages have better sterilization effect than antibiotics in a high concentration of bacteria, and that, collaboration of phages and antibiotics have better sterilization effect than alone of phages or antibiotics in a low concentration medium of bacteria ( Fig. 9,10,11).
Phylogenetically of Salmonella paratyphi mA reveal that has a high similarity and correlation with lytic spectrum of phage KM16. Similar results were found that tail ber adhesion features was a rare polyglycine rich domain for host recognition of Salmonella phage S16 [33]. Furthermore, the de cient outer core of lipopolysaccharides was proposed for Escherichia coli O157:H7 KIT03 recognise and infect host bacteria [34].
The ability of Salmonella to form bio lms on different food surfaces increases the risk of crosscontamination, particularly in poultry products, which was a serious problem for food industries and public health [35][36][37]. Although the signi cant problems in pathogen control caused by bio lms, exploiting effective eliminate of bio lms is still challenging [38]. Until now, there is no ideal technology of bio lm control, hence, the new control strategies for bio lm are constantly recommended [39].  [44].
The result shown that phage KM16 and kanamycin sulfate can infect Salmonella bio lm and has the potential to reduce tested Salmonella paratyphi A NA3 strains. Antibiotics have better anti-bio lm effect than phages in a low concentration medium of bacteria (Fig. 9,10,11). Nonetheless, phages have better anti-bio lm effect than antibiotics in a high concentration of bacteria, and that, collaboration of phages and antibiotics have better anti-bio lm effect than alone of phages or antibiotics in a low concentration medium of bacteria ( Fig. 9,10,11). The data of this study provided the strong evidence that the application of phage could reduce the growth and bio lm of Salmonella that are important to maintain public health.

Conclusions
In this work, we have found the host speci c lytic effect of Salmonella paratyphi phage correlation with tertiary structure of Salmonella pili, which this research provided a new perspective to understand the relationship between phage lytic spectrum and difference of host strains, this is important in eld of phage lytic spectrum knowledge. Collaboration of phage KM16 and kanamycin sulfate have better antibio lm effect than alone of KM16 and kanamycin sulfate in low concentration of bacterial culture, KM16 has better anti-bio lm effect than kanamycin sulfate in high concentration of bacterial culture. The data of this study provided a strong evidence of application phage to reduce the growth of Salmonella bio lm of which were important for public health.

Bacterial strains and growth conditions
Salmonella paratyphi A NA3 was isolated from the patient in Yunnan rst people's hospital, China, and it was used as host for phage isolation. The host strains were grown aerobically on LB plates or in LB broth (Difco, Detroit, MI, USA) that incubated at 37 ℃. Soft top agar containing LB broth was prepared with 0.5% agar (M/M) for phage plaque con rmation and LB agar plates were prepared with broth that supplemented with 1.8% agar (M/M). All strains of Salmonella stock cultures were stored at −80 ℃ of LB broth (Difco, Detroit, MI, USA) which containing 20% (V/V) glycerol.

Phage isolation and puri cation
Phages were isolated from residential sewage sump and pig slaughterhouse sump samples collected from Kunming city, China in September 2018. The method of isolate phages was according to the following methods with brief modi ed [45]. Brie y, 10 g of each sample was mixed with 20 mL sterile normal saline (0.9% NaCl) buffered in 50 mL sterile centrifuge tube and then shake for 2 h using incubator with 200 rpm at room temperature. Then, samples were centrifuged at 5000×g for 15 min and ltered using 0.22 µm lter membrane. 10 mL of each ltering medium was added to 30 mL of LB broth containing the 1% of overnight culture of the host strain and then incubated for 48 h. After that, Cultures were centrifuged at 8 000×g for 15 min and the supernatant was ltered using 0.22 µm lter membrane. The ltrate was diluted 10 times in series and mixing in 5 mL of molten 0.5% LB soft agar containing Salmonella (2×10 8 cfu/mL), and immediately add to LB plate that containing 1.8 agar. Overnight culture and plaque formation was observed. Single phage plaque was selected for phage puri cation and repeat three times.
The thermotolerance, optimum pH, optimum MOI, growth curve and transmission electron microscopy (TEM) of isolated phages Thermotolerance The phage stock was diluted to 1 × 10 8 pfu/mL with LB broth. Placed of 1 mL diluted phage in temperature controller of 4 ℃, 25 ℃, 37 ℃, 42 ℃, 50 ℃, 60 ℃ and 90 ℃ for 1 h, respectively. Following determination of phage titer at different temperature. The experiment was repeated three times.

Optimum MOI
Multiplicity of infection (MOI) was the ratio of the number of phages added to the number of host bacteria at the time of initial infection. According to the MOI of 0.0001, 0.001, 0.01, 0.1 1, 10 and 100 added phage stocks that diluted by LB broth into 10-fold series. Aliquots of each phage stocks were mixed with Salmonella paratyphi A NA3 cultures at 10 8 cfu/ml to each ratio and were shocked culture at 37 ℃ for 8 h. The cultures were centrifuged at 10 000×g for 15min at 4 ℃, then the supernatant was ltered by 0.22 µm lter to obtain the phage increment solution, nally the titer of phage increment solution was determined through double plate method, the experiment was repeated three times.

Adsorption rate and growth curve
For adsorption rate measure, co-culture of phage KM16 (1×10 8 pfu/mL) and logarithmic phase host bacterial suspension at the MOI of 0.1, mixing uniformity and adsorbed at 15 ℃, sampling 100 µL at 0, 2, 4, 6, 8, 10, 15, 20 and 30 min, respectively. The supernatant was taken after centrifugation of 12 000 g/min for 5 min. The titer of phage was determined by double -plate method. For growth curve measure, added phage KM16 of 1×10 8 pfu/mLto LB culture containing 1/250 seed uid of host bacteria according the optimum MOI and shake culture at 37 ℃, intermittent sampling was used to determine the titer of the phage.

Transmission electron microscopy
The morphology of the phages particles was analyzed by transmission electron microscopy (TEM).
Brie y, phage stock dilution (approximately 2×10 8 to 2×10 9 pfu/mL) was deposited on copper grids with carbon-coated formvar lms, stained with 2% uranyl-acetate (pH 4.0). Phage samples were imaged using a Philips EM 300 electron microscope, operated at 80 kV at the Jiangnan university (Wuxi, China). Phages were classi ed and identi ed referring to the International Committee on Taxonomy of Viruses [46].

Phage genomic DNA extraction, sequencing and bioinformatics analysis
Phage genomic extraction and restriction enzyme digestion Firstly, phage was puri ed from concentrated to a high titer stock using 10 kDa lter (about 10 9 to 10 10 ). Puri ed phages were treated with RNase and DNase at 37 ℃ for 1 h. Then, Takara minibest viral RNA/DNA Extraction kit (Cat#9766) was carried out to obtain puri ed phage genomic DNA. Restriction endonuclease EcorI, Hind III, Not I and Xhol I were used for phage genome digestion, respectively.

Sequencing and bioinformatics analysis
Extracted phage genomic DNA was sequenced using a Illumina Hiseq (Sangon Biotech, China). The original sequencing data were evaluated by FastQC and assembled with SPAdes assembler software. The NCBI Blast compare with multiple databases of CDD, KOG, COG, NR, NT, PFAM, Swissprot and TrEMBL were used for function annotation information of gene protein sequence.

Host-range determination
The host range of the phage KM16 were determined by the spot test method [47]. The isolated strains were tested for susceptibility of phage KM16 ( ten strains of Salmonella paratyphi A and two strains of Salmonella paratyphi B). Generally, each of 200 uL reference strains (10 9 cfu/mL) was added to 5 mL of lique ed LB soft agar (LB broth with 0.5% agar), and poured over the LB 1.8% agar plate. Three minutes later, single drops of phage suspension were added and incubated at 37 ℃ for 24 h.

Phylogenetic and genetically speci c analysis of host
The genome of host Salmonella paratyphi were extracted by Takara minibest bacteria genomic DNA extraction kit. Then sequencing of 16S gene was performed with 9F upstream and 1512R downstream primers (F:GAGTTTGATCCTGGCTCAG; R:ACGGHTACCTTGTTACGACTT) in Sangon, China. Then biofilm and crisp-cas related gene sequencing was performed with primers in Sangon, China ( Table 3) .The phylogenetic analysis of 16S, biofilm and crisp-cas related gene of Salmonella paratyphi were performed using Molecular Evolutionary Genetics Analysis (MEGA7.0) software. The gene speci city of biofilm and crisp-cas related gene was studied using the software of Boiedit.
Homology modelling of Salmonella mA (pili) protein Homology modelling of Salmonella mA (pili) protein tertiary structure was performed using SWISS-MODEL online suit. The tertiary structure of Salmonella mA among Salmonella paratyphi A-A, Salmonella paratyphi A-NA3 and Salmonella paratyphi B-H were predicted.   Plaques formed by phage KM16, the host strains of Salmonella paratyphi A NA3 after an overnight incubation at 37℃.

Figure 3
Morphological features of phage KM16 with host of Salmonella paratyphi A NA3 by transmission electron microscopy (TEM).

Figure 5
Restriction enzyme digests of phage KM16. Phage KM16 DNA was digested by EcoRI, Hind III, NotI and Xhol I.

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