Controlling Multidrug-Resistant Staphylococcus aureus by Combined Use of Antimicrobials and Phage STPX-6 with Broad Spectrum and High Efficiency

Abstract

Objective:

The emergence of antibiotic-resistant Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), poses a great challenge for animal and public health. This study aimed to isolate a broad-spectrum and high-efficiency MRSA phage and explore the phage–antibiotic synergistic effect on MRSA.

Results:

Phage STPX-6 belongs to Caudovirales, Podoviridae. It has a hexahedral head and a short tail. Its genome length was 17,007 bp, and it did not contain resistance genes and virulence genes. STPX-6 lysed 79.6% (133/167) of 167 S. aureus and 87.96% (95/108) of MRSA from different sources. The titer of phage was 1.18 × 10¹⁰ PFU/mL, the optimal multiplicity of infection was 1, the latent period and lysis period were about 10 min and 60 min, respectively, and the burst amount was 68 PFU/cell. At 50°C and 70–90°C, the titer of STPX-6 was maintained at about 10¹⁰ PFU/mL and at least 10³ PFU/mL, respectively. In the range of pH 4–12, the titer of phage remained above 10⁸ PFU/mL, and it remained above 10⁴ PFU/mL at pH 2, 3, 13, and 14. The combined application of phage STPX-6 and enrofloxacin, doxycycline, ampicillin could reduce the minimum inhibitory concentration (MIC) of the three antibiotics to 1/4 MIC, 1/16 MIC, and 1/2 MIC, respectively.

Conclusion:

This study found that for the host MRSA, lytic phage STPX-6 had the characteristics of a broad lytic spectrum, a short latent period, strong adaptability and strong tolerance to high temperature, a strong acid and strong alkali environment, and might maintain certain activity under extreme environment. More importantly, the combination of phage STPX-6 with enrofloxacin, doxycycline, and ampicillin could reduce the antibiotic concentration used for MRSA. In other words, phages as new antibacterial agents have received increasing attention. The combined application of phages and antibiotics provides a new method for controlling multidrug resistant bacteria and reduce the use of antibiotics.

Keywords

phage whole-genome sequence MRSA

Introduction

In recent years, with the continuous expansion of the scale of the livestock and poultry breeding, the widespread and imprudent use of antimicrobials has become a global problem. It not only leads to excessive antibiotic residue in animal-origin food but also causes food safety problems. What’s more, the antibiotic resistance rates of poultry is rising, and the resistance spectrum is also expanding. Since the 1960s, with the clinical use of methicillin, methicillin-resistant Staphylococcus aureus (MRSA) have been detected in animal products in several countries, including Asia, Europe, and North America (Andie et al., 2018). Studies have shown that MRSA strains were resistant to almost all β-lactam antibiotics and were highly resistant to other antibiotics (Sharon and Gavin, 2015). In addition, MRSA generally has a strong ability to adapt to the environment and colonization. It is worth noting that MRSA usually leads to high infection and mortality rates (Vanessa et al., 2023). However, there are few effective therapeutic drugs in clinical settings (Paul et al., 2018), and the treatment of MRSA has become a serious challenge. Therefore, it is urgent to find a new option to effectively control MRSA from the source.

It is well known that phages, as the new antibacterial agents, have attracted widespread attention due to their advantages of safety, no residue, and strong specificity (Wernicki et al., 2017). However, studies have confirmed that bacteria could adapt to phages and produce resistance, which indicated that it was very important to overcome the resistance of bacteria to phages (Tania et al., 2014). Therefore, phage–antibiotic combination therapy has great potential (Kebriaei et al., 2020; Kebriaei et al., 2022; Razieh et al., 2023). In this study, the dominant MRSA strain ST9 was used as the host strain to study the combined application effect of phage STPX-6 with enrofloxacin, doxycycline, or ampicillin, which could be an effective preventive measure in MRSA.

Materials and Methods

Strain source

The host MRSA strain MRA202109P0009 used for phage screening was isolated from a healthy broiler farm in Shandong Province, China, in 2021 (Xiao et al., 2023), which was resistant to 13 types of antibiotics according to the Clinical and Laboratory Standards Institute (CLSI) and contained 12 drug resistance genes. The laboratory growth conditions of the host MRSA strain were: Tryptone Soya Agar (TSA) culture medium at 37°C for 18–20 h or 180 r/min LB broth at 37°C for 6 h. A total of 167 strains of S. aureus were isolated from healthy poultry (layer, broiler, waterfowl farm) by throat swabs from 49 farms in 2021 (Xiao et al., 2023). They were used to verify the bactericidal effect and determine the lytic spectrum of phage. Supplementary Table S1 shows the information of different isolates of S. aureus strains and the host strain.

Isolation, purification, host range determination, and physiological characteristics of phage

According to Fang Tang (Tang et al., 2019), phage STPX-6 was isolated and purified from the broiler fecal samples in Shandong Province, China, in 2021. In total, 5 g of fecal samples were collected for sample treatment. Phages were isolated and purified by plaque assay and double-layer agar assay until the uniform size of the phage plaque appeared. After purification, phages were added to SM buffer solution and stored temporarily at 4°C. The titer of phage was determined by double-layer agar assay.

A total of 167 S. aureus isolates were used to determinate the host range of phage STPX-6 (Tang et al., 2019). The transmission electron microscope (TEM) observation, optimal multiplicity of infection (MOI) determination, one-step growth curve and burst amount determination, thermal stability and pH stability determination of phage were conducted as described previously (Tang et al., 2019).

The whole-genome sequencing of STPX-6 was conducted by Shanghai Tappu Biotechnology Co., LTD through Illumina (NovaSeq 6000) sequencing platform, SPAdes and MEGAHIT software. The genome map of phage STPX-6 was drawn by Phastest software. The BLAST-ORFfinder programs of the National Center for Biotechnology Information website and Phastest software were used for preliminary analysis and gene annotation of the coding sequence of phage STPX-6 genome.

Combined antibacterial experiments of bacteriophage and antibiotics

According to CLSI, the minimum inhibitory concentrations (MICs) of enrofloxacin, doxycycline, and ampicillin to host MRSA strain were determined by broth micro dilution method. Antibiotics with different concentrations were added to the bacteriophage STPX-6 solution for 16 h, and the effect of each antibiotic on phage titer was observed by double-layer agar assay.

The combined antibacterial experiment was divided into four groups: blank control group, antibiotic group, bacteriophage group, and combined application group. According to the MOI, the initial concentrations of host bacteria and phage were set at 5 × 10⁵ CFU/mL, respectively. To observe whether there was phage–antibiotic synergy, the bacterial concentration in each group was measured every 4 h by the spread plate counting method and was monitored for 20 h in total. Three independent experiments were repeated.

Statistical analyses

Statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA) and Origin2019 (OriginLab Corporation, USA). SD was used for the analysis of the degree of dispersion in data points. Statistical significances were established at *, ** and *** and were represented by p < 0.05, p < 0.01, and p < 0.001, respectively.

Results

Isolation, purification, and titer determination of phage

In this study, MRSA strain MRA202109P0009 was used as the host bacteria. The phage named STPX-6 was isolated from the feces of broiler chickens. The morphology of phage plaque in the double-layer agar assay was shown in Figure 1. The phage plaque formed by STPX-6 was 2∼3 mm in diameter, and the inside was smooth and clear. When the purified phage solution was coated on TSA plate alone, no bacterial growth was observed. The titer was 1.18 × 10¹⁰ PFU/mL.

FIG. 1.

Morphology of phage plaque of STPX-6.

Host range analysis of STPX-6

Table 1 and Supplementary Table S1 showed that STPX-6 lysed 79.6% (133/167) of S. aureus and 87.96% (95/108) of MRSA. In addition, the lytic effect of STPX-6 on three different sources of S. aureus was different. In particular, the lytic rates of STPX-6 on broiler strains, laying hens strains and waterfowl strains were 78.95% (60/76), 81.48% (44/54), and 78.38% (29/37), respectively.

Table 1.

Host Range Analysis of STPX-6

S. aureus Source	−	+	++	+++	Lytic Rate
Broiler	21.05% (16/76)	9.21% (7/76)	19.74% (15/76)	50% (38/76)	78.95% (60/76)
Laying hens	18.52% (10/54)	7.41% (4/54)	22.22% (12/54)	51.85% (28/54)	81.48% (44/54)
Waterfowl	21.62% (8/37)	13.51% (5/37)	13.51% (5/37)	51.35% (19/37)	78.38% (29/37)

Note: The morphology of phage plaque is described as follows: “+++” the degree of phage plaque transparency = 100%; “++” 50% ≤ the degree of phage plaque transparency < 100%; “+” 0 < the degree of phage plaque transparency < 50%; “−” no plaque.

Physiological characteristics of STPX-6

Morphological observation of phage under TEM

TEM (Fig. 2) showed that phage STPX-6 belonged to Caudovirales, Podoviridae. Its outer capsid was bright and the inner nucleic acid was dark. The head had a standard regular hexahedron structure with a diameter of about 40 nm. It had a nonshrinkable tail of about 20 nm.

FIG. 2.

Transmission electron microscopic morphology of phage STPX-6.

MOI of phage STPX-6

As shown in Table 2, when the MOI of STPX-6 was 1, the offspring of STPX-6 infected with host bacteria had the highest titer, reaching 6.35 × 10¹⁰.

Table 2.

Optimal Multiplicity of Infection of Phage STPX-6

Multiplicity of infection	Phage concentration (PFU/mL)	Host bacteria concentration (CFU/mL)	Phage titer 5 h after infection
Multiplicity of infection	Phage concentration (PFU/mL)	Host bacteria concentration (CFU/mL)	Phage titer 5 h after infection	0.001	1 × 10⁵	1 × 10⁸	1.43 × 10⁸
0.01	1 × 10⁶	1 × 10⁸	1.7 × 10¹⁰
0.1	1 × 10⁷	1 × 10⁸	4 × 10⁹
1	1 × 10⁸	1 × 10⁸	6.35 × 10¹⁰
10	1 × 10⁹	1 × 10⁸	3.15 × 10⁹

One-step growth curve of phage STPX-6

As shown in Figure 3, the latent period of the phage STPX-6 was approximately 10 min and the titer increased rapidly between 10 min and 70 min. The lysis period was approximately 60 min. The titer basically remained at about 10¹¹ PFU/mL after 70 min, and the burst amount was approximately 68 PFU/cell.

FIG. 3.

One-step growth curve of STPX-6.

Thermal stability and pH stability of phage STPX-6

The result of the thermal stability of phage STPX-6 was shown in Figure 4A. At 50°C, the titer of STPX-6 hardly changed, but when the temperature rose to 70°C or higher and lasted for 20 min, the titer decreased significantly. After being treated at 90°C for 60 min, phage STPX-6 still maintained a certain bactericidal activity, indicating that STPX-6 had a high temperature resistance.

FIG. 4.

Thermal stability and pH stability of STPX-6. (A) Effect of different temperature on the titer of STPX-6. (B) Effect of different PH on the titer of STPX-6.

In order to further study the pH stability of STPX-6, its titer at different pH was determined (Fig. 4B). In the pH range of 6–12, the titer of STPX-6 did not change significantly. In the range of pH 4–5, the titer of STPX-6 decreased slightly. When pH value was lower than 3, the titer of STPX-6 decreased significantly, and when pH value was 1, the phage STPX-6 was completely inactivated. Although the titer of STPX-6 decreased significantly in the pH range of 13–14, some bacteriophages still survived in the alkaline condition of pH 14. These results indicated that phage STPX-6 had strong tolerance to acidic and alkaline environments, especially alkaline environments.

Genomic features of phage STPX-6

Whole-genome sequencing analysis (Fig. 5) showed that STPX-6 was encoded by double-stranded DNA, with a total genome length of 17,007 bp and a GC% content of 29.65% (GenBank accession number: PP723060). According to the search results of ORF finder, STPX-6 encoded 78 open reading frames (ORFs) larger than 78 bp, of which 38 were positive chains and 40 were negative chains (Table 3). Analysis showed that 20 ORFs had the functions of coding genes, including main structural modules, DNA replication and assembly modules, lysis modules, and other hypothetical proteins (unknown functional proteins). No genes related to antibiotic resistance and virulence factors of bacteria were found.

FIG. 5.

Genome map of STPX-6.

Table 3.

Open Reading Frame Analysis of the Phage STPX-6 Genome

ORFs	Strand	Start	Stop	Length (nt/aa)	Function
ORF1	+	115	525	411 \| 136	Hypothetical protein
ORF2	+	538	720	183 \| 60	Hypothetical protein
ORF3	+	727	1953	1227 \| 408	Head protein
ORF4	+	3106	3189	84 \| 27	Hypothetical protein
ORF5	+	3421	3522	102 \| 33	Hypothetical protein
ORF6	+	3712	5652	1941 \| 646	Minor structural protein
ORF7	+	5860	5943	84 \| 27	Hypothetical protein
ORF8	+	7468	9231	1764 \| 587	Major Tail protein
ORF9	+	9967	10,044	78 \| 25	Hypothetical protein
ORF10	+	10,510	10,719	210 \| 69	Hypothetical protein
ORF11	+	10,963	11,073	111 \| 36	Hypothetical protein
ORF12	+	11,092	11,223	132 \| 43	Hypothetical protein
ORF13	+	12,118	12,204	87 \| 28	Hypothetical protein
ORF14	+	13,081	13,161	81 \| 26	Hypothetical protein
ORF15	+	1682	1762	81 \| 26	Hypothetical protein
ORF16	+	1823	1903	81 \| 26	Hypothetical protein
ORF17	+	1967	2950	984 \| 327	Upper collar protein
ORF18	+	3809	3889	81 \| 26	Hypothetical protein
ORF19	+	4946	5053	108 \| 35	Hypothetical protein
ORF20	+	6479	7408	930 \| 309	Tail fiber
ORF21	+	8423	8512	90 \| 29	Hypothetical protein
ORF22	+	9233	9655	423 \| 140	Holin
ORF23	+	11,495	11,623	129 \| 42	Hypothetical protein
ORF24	+	11,879	12,031	153 \| 50	Hypothetical protein
ORF25	+	12,956	13,042	87 \| 28	Hypothetical protein
ORF26	+	14,705	14,845	141 \| 46	Hypothetical protein
ORF27	+	15,056	15,157	102 \| 33	Hypothetical protein
ORF28	+	16,217	16,300	84 \| 27	Hypothetical protein
ORF29	+	2532	2627	96 \| 31	Hypothetical protein
ORF30	+	2922	3698	777 \| 258	Lower collar protein
ORF31	+	5664	6416	753 \| 250	Endolysin
ORF32	+	6684	6776	93 \| 30	Hypothetical protein
ORF33	+	9630	11,069	1440 \| 479	Putative tail lysin
ORF34	+	11,607	11,717	111 \| 36	Hypothetical protein
ORF35	+	12,672	12,779	108 \| 35	Hypothetical protein
ORF36	+	13,389	13,469	81 \| 26	Hypothetical protein
ORF37	+	14,916	15,017	102 \| 33	Hypothetical protein
ORF38	+	15,432	15,518	87 \| 28	Hypothetical protein
ORF39	−	16,644	16,342	303 \| 100	Hypothetical protein
ORF40	−	16,266	16,189	78 \| 25	Hypothetical protein
ORF41	−	14,730	13,483	1248 \| 415	DNA encapsidation protein
ORF42	−	13,467	11,182	2286 \| 761	DNA polymerase
ORF43	−	10,974	10,840	135 \| 44	Hypothetical protein
ORF44	−	10,722	10,645	78 \| 25	Hypothetical protein
ORF45	−	10,329	10,225	105 \| 34	Hypothetical protein
ORF46	−	9492	9274	219 \| 72	Hypothetical protein
ORF47	−	6585	6349	237 \| 78	Hypothetical protein
ORF48	−	6177	6079	99 \| 32	Hypothetical protein
ORF49	−	5970	5878	93 \| 30	Hypothetical protein
ORF50	−	5736	5572	165 \| 54	Hypothetical protein
ORF51	−	3435	3322	114 \| 37	Hypothetical protein
ORF52	−	2625	2548	78 \| 25	Hypothetical protein
ORF53	−	2529	2443	87 \| 28	Hypothetical protein
ORF54	−	16,409	16,332	78 \| 25	Hypothetical protein
ORF55	−	16,064	15,696	369 \| 122	Single stranded DNA-binding protein
ORF56	−	15,260	14,778	483 \| 160	Hypothetical protein
ORF57	−	13,313	13,197	117 \| 38	Hypothetical protein
ORF58	−	11,906	11,829	78 \| 25	Hypothetical protein
ORF59	−	11,468	11,376	93 \| 30	Hypothetical protein
ORF60	−	11,321	11,229	93 \| 30	Hypothetical protein
ORF61	−	5297	5130	168 \| 55	Hypothetical protein
ORF62	−	4145	4026	120 \| 39	Hypothetical protein
ORF63	−	16,840	16,637	204 \| 67	Hypothetical protein
ORF64	−	16,324	16,088	237 \| 78	Hypothetical protein
ORF65	−	15,787	15,683	105 \| 34	Hypothetical protein
ORF66	−	15,646	15,467	180 \| 59	Hypothetical protein
ORF67	−	15,457	15,263	195 \| 64	Hypothetical protein
ORF68	−	15,145	15,065	81 \| 26	Hypothetical protein
ORF69	−	14,797	14,651	147 \| 48	Hypothetical protein
ORF70	−	10,678	10,415	264 \| 87	Hypothetical protein
ORF71	−	10,297	10,148	150 \| 49	Hypothetical protein
ORF72	−	9982	9893	90 \| 29	Hypothetical protein
ORF73	−	5557	5471	87 \| 28	Hypothetical protein
ORF74	−	5020	4943	78 \| 25	Hypothetical protein
ORF75	−	3844	3734	111 \| 36	Hypothetical protein
ORF76	−	3718	3569	150 \| 49	Hypothetical protein
ORF77	−	2443	2342	102 \| 33	Hypothetical protein
ORF78	−	1303	1214	90 \| 29	Hypothetical protein

Combined antibacterial effect of phage STPX-6 and three antibiotics

An increasing number of studies have shown that the combination of phages and antibiotics is more effective in controlling pathogenic bacteria than either alone. This could better control the existence of the respective resistant bacteria and produce a phage–antibiotic synergy, achieving a better antibacterial effect (Clara and Michael, 2016). Next, we conducted a bacteriostatic experiment of STPX-6 combined with enrofloxacin, doxycycline, and ampicillin, which were commonly used in animal clinical for S. aureus. First, the MICs of enrofloxacin, doxycycline, and ampicillin against host bacteria were determined, which were 8 µg/mL, 8 µg/mL, and 32 µg/mL, respectively. According to the CLSI standard, the host bacteria was resistant, intermediate, and resistant to the above three antibiotics, respectively. Then, the effects of three antibiotics on the titer of STPX-6 were studied. Compared with the control group, the titer of STPX-6 after interacting with antibiotic for 16 h at the concentration of MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC, and 1/16 MIC, and there was no significant difference (p > 0.05). Therefore, these three antibiotics had no effect on the titer of STPX-6.

Combined antibacterial effect of phage STPX-6 and enrofloxacin

In order to further explore the bactericidal effect of the combination of enrofloxacin and phage STPX-6, a phage–antibiotic synergistic experiment was carried out, and the results were shown in Figure 6A. In the blank group, the bacteria grew normally from 0 to 20 h, and the final concentration reached 2.67 × 10⁸ CFU/mL. When enrofloxacin with different concentrations (1/2 MIC, 1/4 MIC, 1/8 MIC, and 1/16 MIC) was used alone, the bacterial concentration did not decrease significantly. At the same time, the bacteriostatic effect of phage group alone was the best within 4 h, and it gradually decreased from 4 h to 20 h, and the bacterial concentration reached 1.17 × 10⁷ CFU/mL at 20 h. Interestingly, the combined application group showed that different concentrations of enrofloxacin and phage STPX-6 could effectively kill all bacteria at 4–12 h. It is worth noting that the combination of phage STPX-6 and enrofloxacin at 1/8 MIC and 1/16 MIC had significant bacteriostatic effect at 4–12 h (p < 0.001). After 12 h, the bacteriostatic effect decreased, and the bacterial concentration was 2.13–2.17 log₁₀ CFU/mL lower than that of the blank group. Last but not least, the combination of MIC, 1/2 MIC, and 1/4 MIC concentration of enrofloxacin and phage STPX-6 had a strong synergistic effect, and there was no bacterial growth within 0–20 h.

FIG. 6.

Bactericidal results of combination of STPX-6 and three antibiotics. (A–C) Bactericidal results of combination of STPX-6 and enrofloxacin, doxycycline, and ampicillin.

Combined antibacterial effect of phage STPX-6 and doxycycline

It is well known that doxycycline belongs to tetracycline antibiotics, which have a broad antibacterial spectrum and good clinical effect. In this study, the combined bactericidal effect of doxycycline and phage STPX-6 was further explored, and its synergistic effect was shown in Figure 6B. As can be seen from the figure, bacteria in the blank group grew normally with 0–20 h, and the final concentration reached 2.5 × 10⁸ CFU/mL. When different concentrations of doxycycline (1/2 MIC, 1/4 MIC, 1/8 MIC, and 1/16 MIC) were used alone, the bacterial concentration did not decrease. At the same time, the bacteriostasis effect of phage group was the best within 4 h, and it gradually decreased from 4 h to 20 h, the bacterial concentration reaching 6.87 × 10⁷ CFU/mL at 20 h. The combined application group showed that the concentration of 1/16 MIC doxycycline and phage STPX-6 maintained a good synergistic bactericidal effect within 4–20 h (p < 0.001), and no bacterial growth was observed. In addition, the concentration of 1/8 MIC doxycycline also had a good synergistic bactericidal effect with phage STPX-6 within 8–16 h (p < 0.001), and the bacterial concentration at 20 h was 2.99 log₁₀ CFU/mL lower than that of blank control group, showing a good synergistic bactericidal effect (p < 0.001). However, there was no synergistic relationship between doxycycline and bacteriophage STPX-6 at higher concentrations (MIC, 1/2 MIC, and 1/4 MIC), which may be due to the antagonism between doxycycline and STPX-6.

Combined antibacterial effect of phage STPX-6 and ampicillin

Ampicillin, a beta-lactam antibiotic, is used to treat a variety of bacterial infections. As shown in Figure 6C, the synergistic effect of ampicillin and phage STPX-6 was studied. In the blank control group, the bacteria grew normally from 0 h to 20 h, and the final concentration reached 1.52 × 10⁸ CFU/mL. As shown in the figure, when different concentrations of ampicillin (1/4 MIC, 1/8 MIC, and 1/16 MIC) were used alone, bacterial concentration did not decrease significantly. According to the data of phage group, the bacteriostasis effect was the best within 4 h and gradually decreased within 4–20 h. However, it was worth noting that the MIC and 1/2 MIC concentration of ampicillin in the combined application group had a significant synergistic bactericidal effect with the phage combination (p < 0.001), and no bacterial growth was observed in all periods. In addition, a synergistic bactericidal effect was also observed after 1/4 MIC ampicillin interacted with phage STPX-6 for 20 h (p < 0.001). After 20 h of treatment, compared with the blank group, the bacterial concentration in 1/8 MIC and 1/16 MIC phage groups finally decreased by 2.94 log₁₀ CFU/mL and 2.81 log₁₀ CFU/mL, respectively.

In summary, when phage STPX-6 was 5 × 10⁵ PFU/mL and the minimum concentrations of enrofloxacin, doxycycline and ampicillin could be reduced to 1/4 MIC, 1/16 MIC, and 1/2 MIC, respectively, and the phage–antibiotic combination group had an obvious combined bactericidal effect.

Discussion

S. aureus is the third major pathogen causing bacterial food-borne diseases (Di Pinto et al., 2004). The World Health Organization (WHO) listed it as a high-priority pathogen (Subramanya et al., 2021). As we all know, with the widespread use of antibiotics worldwide, the antibiotic resistance of S. aureus has become increasingly serious in recent years, especially the multidrug-resistant strains typical of MRSA have been increasingly reported. According to the Centers for Disease Control and Prevention (CDC), approximately 241,000 people in the United States were sickened each year by consumption of animal-derived foods contaminated with S. aureus (Zeaki et al., 2019), among which, about 80,000 cases of MRSA occurred every year, resulting in about 11,000 deaths (Kochanek et al., 2016). In recent years, with the rise and application of the concept of “One Health,” many studies have found that human infection with S. aureus is more and more closely related to animals (Hanning et al., 2012; Haag et al., 2019). Therefore, MRSA, in animal-derived foods, would not only bring huge economic losses to animal husbandry due to difficult treatment but also be transmitted to people through the food chain, which seriously threatens human health. At present, the research and development of anti-drug products has become an important breakthrough to solve the serious resistance of MRSA. Recent studies have shown that the value of phage therapy as a potential adjuvant to traditional antibiotics has been re-recognized (Ferry et al., 2020; Jault et al., 2019). It has shown good application effects (Adhya et al., 2014; Cai et al., 2019; Wei et al., 2020; Wernicki et al., 2017) in human clinics, disinfection of livestock and poultry breeding environments, prevention and treatment of bacterial infection, and so on, and it has gradually become the focus of attention in recent years. Poultry, as the source of common animal-derived food, is an important host of S. aureus. In this study, a virulent phage was isolated from feces samples of poultry farms in Shandong Province, China, and its lysis effect on S. aureus isolated from different poultry was evaluated, so as to control the spread of food-borne MRSA from the breeding process as the source. Through TEM observation, it was found that STPX-6 belonged to Caudovirales, Podoviridae, which had a broad lytic spectrum and high efficiency for the host MRSA strain. Importantly, STPX-6 had a certain ability to lyse S. aureus from different poultry sources, and lysed 79.6% (133/167) and 81.48% (44/54) of S. aureus and S. aureus from laying hens, respectively. In contrast, the phage isolated by Wang Yifan et al. (Wang et al., 2022) could only lyse the host bacteria, but it had no ability to lyse other experimental strains of S. aureus, and its lytic spectrum was narrow.

In the natural environment, it is well known that the ability of phage to infect host bacteria depends on its tolerance to environmental pressure. Therefore, good tolerance to environmental pressure is a prerequisite for phages to be used as new biological antibacterial agents. In this study, the biological characteristics and genome-wide sequencing results showed that for the host MRSA, phage STPX-6 had the characteristics of short latent period, high burst amount, strong tolerance to high temperature, strong acid, and strong alkaline environment, and maintained certain activity under extreme environment, indicating its potential application value as a candidate for phage preparation. Compared with the research results of Cai Tianshu et al. (Cai et al., 2013), phage STPX-6 showed better thermal stability. For example, some STPX-6 were still alive under extreme conditions of 90°C. However, when the temperature is higher than 70°C, phage activity was unstable. Carey-Smith GV (Carey-Smith et al., 2006) concluded that most phages could maintain high activity at pH 5–9, whereas phage STPX-6 could still maintain partial activity at pH 2 and 14, which indicated that phage STPX-6 was still useful in extreme pH environments. In this study, the burst amount of phage STPX-6 was slightly lower than that of S. aureus phage (about 80 PFU/cell) isolated by Fan Jindai et al. (Fan et al., 2015), but it was higher than that of S. aureus phage found by Dang Ruiying (Dang et al., 2022), Cai Tianshu (Cai et al., 2013), Jéssica Duarte da Silva (Jéssica et al., 2023) and Peisong Zhao et al. (Zhao et al., 2024). In addition, the latent period of STPX-6 was only 10 min, which was much shorter than that of other S. aureus phages studied by Jéssica Duarte da Silva (Jéssica et al., 2023) and Peisong Zhao (Zhao et al., 2024). Therefore, these biological characteristics indicated that for the host S. aureus, phage STPX-6 had the characteristics of short latent period, high burst amount, and easy mass preparation, which had great advantages and potential for the prevention and control of MRSA.

Phage is a virus that infects bacteria in a special host. Because of its high affinity and rapid proliferation, it is an important anti-infection application in the initial stage (Luo et al., 2018; Macneal et al., 1934; Zhang et al., 2017). It is worth noting that a large number of studies have proved that bacteria could also develop resistance to phage in the process of using it to treat bacterial infections (Liu et al., 2021; Luo et al., 2022). Therefore, the combination of bacteriophage and antibiotics can better control the respective resistant bacteria and produce a phage–antibiotic synergistic effect, thus better reducing the number of bacteria (Clara and Michael, 2016; Ma et al., 2017; Rahman et al., 2011). The earliest report of this kind of research in China could be traced back to the 1950s. Peng Renling (Peng, 1959) found that the combined application of chloramphenicol and phage had a good synergistic effect on typhoid bacillus. Mathias Jansen et al. (Mathias et al., 2018) found that KARL-1, a new T4-like phage, had a synergistic antibacterial effect on multidrug resistant Acinetobacter baumannii when used in combination with various antibiotics. Phage YC#06 isolated and identified by Luo Jun (Luo et al., 2022) against highly multidrug-resistant Acinetobacter baumannii had a synergistic effect with antibiotic mixtures such as chloramphenicol, imipenem, and cefotaxime. In the early stage of this study, the combined application of phage and antibacterial drugs was explored. A variety of antibiotics commonly used in veterinary clinics and with serious drug resistance were selected for relevant experiments. The results showed that only enrofloxacin, doxycycline, and ampicillin had different degrees of synergy with bacteriophage STPX-6 and could reduce the MIC of antibacterial drugs. In this study, when the concentration of phage STPX-6 was 5 × 10⁵ PFU/mL, the combined treatment group had obvious combined antibacterial effect, whereas the concentrations of enrofloxacin, doxycycline, and ampicillin could be reduced to 1/4 MIC, 1/16 MIC, and 1/2 MIC, respectively. It was still necessary to continue to explore the effect of phage–antibiotic synergistic inhibition of S. aureus in vivo in different animals, and we continued to monitor whether phage STPX-6 would produce new drug resistance during the clinical application of animals. The study revealed that the combined application of bacteriophage STPX-6 and antibiotics not only provided support for the research and development of antidrug products for the prevention and control of MRSA but also reduced the use of antibiotics and thus reduced the production of drug-resistant strains, which had important research value and clinical production guidance significance.

In conclusion, the effect of a single antimicrobial agent on the eliminating bacterial infection is extremely limited, and its excessive use will lead to the increasing antibiotic resistance, as well as phage therapy. Therefore, the combined application of phage and antibiotic has become a trend, which can not only reduce the use of antibiotics but also inhibit the emergence of self-resistant bacteria. This provides a feasible scheme for reducing the use of antibiotics in breeding, guiding clinical drug use in livestock and poultry breeding and eradicating new multidrug-resistant strains.

Authors’ Contributions

L.W.: Conceptualization, investigation, methodology, writing—original draft. Z.X.: Conceptualization, methodology, data curation, writing—original draft. J.W.: Conceptualization, validation, formal analysis. N.L.: Methodology, data curation, investigation. W.J.: Supervision, writing—review and editing. Y.L.: Data curation, software, visualization. F.H.: Methodology, data curation, validation. H.L.: Resources, supervision, J.L.: Supervision, resources, writing—review and editing. Z.Q.: Conceptualization, funding acquisition, project administration, resources, supervision, writing—review and editing. J.W.: Funding acquisition, project administration, supervision, Resources.

Footnotes

Funding Information

This study was funded by the National Key Research and Development Program of China (Grant no: 2022YFC2303900).

Disclosure Statement

No Interests to disclose.

Supplemental Material

References

Adhya

, Merril

, Biswas

. Therapeutic and prophylactic applications of bacteriophage components in modern medicine. Cold Spring Harb Perspect Med, 2014; 4(1):a012518; doi: 10.1101/cshperspect.a012518

Andie

, Hermínia

, Javier

, et al. Methicillin-resistant Staphylococcus aureus. Nat Rev Dis Primers, 2018; 31:18033; doi: 10.1038/nrdp.2018.33

Cai

, Wang

, et al. Biological properties and genomics analysis of vB_KpnS_GH-K3, a Klebsiella phage with a putative depolymerase-like protein. Virus Genes, 2019; 55(5):696–706; doi: 10.1007/s11262-019-01681-z

Cai

, Wang

, Lin

, et al. Biological properties of staphylococcus aureus phage and its antibacterial effect in milk. Food Science, 2013; 34(11):147; doi: 10.7506/spkx1002-6630-201311033

Carey-Smith

, Billington

, Cornelius

, et al. Isolation and characterization of bacteriophages infecting Salmonella spp. FEMS Microbiol Lett, 2006; 258(2):182–186; doi: 10.1111/j.1574-6968.2006.00217.x

Clara

, Michael

. Evolutionary rationale for phages as complements of antibiotics. Trends Microbiol, 2016; 24(4):249–256; doi: 10.1016/j.tim.2015.12.011

Dang

, Chang

, Liang

, et al. Isolation and characterization of Staphylococcus aureus phage P82 from dairy mastiti. China Animal Husbandry & Veterinary Medicine, 2022; 49(6):2318–2325; doi: 10.16431/j.cnki.1671-7236.2022.06.032

Di Pinto

, Forte

, Ciccarese

, et al. Comparison of reverse passive latex agglutination test and immunoblotting for detection of staphylococcal enterotoxin A and B. J FOOD SAFETY, 2004; 24(4):231–238; doi: 10.1111/j.1745-4565.2004.00533.x

Fan

, Shen

, Ma

, et al. Isolation, identification and biological characteristics of a bacteriophage of Staphylococcus aureus causing dairy cow mastitis. Chinese Veterinary Science, 2015; 45(12):1236–1241; doi: 10.16656/j.issn.1673-4696.2015.12.005

10.

Ferry

, Batailler

, Petitjean

, et al. The potential innovative use of bacteriophages within the DAC hydrogel to treat patients with knee megaprosthesis infection requiring “debridement antibiotics and implant retention” and soft tissue coverage as salvage therapy. Front Med (Lausanne), 2020; 7:342; doi: 10.3389/fmed.2020.00342

11.

Haag

, Fitzgerald

, Penadés

. Staphylococcus aureus in Animals. Microbiol Spectr, 2019; 7(3); doi: 10.1128/microbiolspec.gpp3-0060-2019

12.

Hanning

, Gilmore

, Pendleton

, et al. Characterization of Staphylococcus aureus isolates from retail chicken carcasses and pet workers in Northwest Arkansas. J Food Prot, 2012; 75(1):174–178; doi: 10.4315/0362-028X.JFP-11-251

13.

Jault

, Leclerc

, Jennes

, et al. Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): A randomised, controlled, double-blind phase 1/2 trial. Lancet Infect Dis, 2019; 19(1):35–45; doi: 10.1016/S1473-3099(18)30482-1

14.

Jéssica

, Luís

, Sílvio

, et al. Genomic and proteomic characterization of vB_SauM-UFV_DC4, a novel Staphylococcus jumbo phage. Appl Microbiol Biotechnol, 2023; 107:7231–7250; doi: 10.1007/s00253-023-12743-6

15.

Kebriaei

, Lev

, Morrisette

, et al. Bacteriophage-antibiotic combinationstrategy: An alternative against methicillin-resistant phenotypes of Staphylococcus aureus. Antimicrob Agents Chemother, 2020; 64(7):e00461-20; doi: 10.1128/AAC.00461-20

16.

Kebriaei

, Lev

, Shah

, et al. Eradication of biofilm-mediated methicillin-resistant Staphylococcus aureus infections in vitro: Bacteriophage-antibiotic combination. Microbiol Spectr, 2022; 10(2):e00411-22; doi: 10.1128/spectrum.00411-22

17.

Kochanek

, Murphy

, Xu

, et al. Deaths: Final data for 2014. Natl Vital Stat Rep, 2016; 65(4):1–122.

18.

Liu

, Zhao

, Hayes

, et al. Overcoming bacteriophage insensitivity in Staphylococcus aureus using clindamycin and azithromycinat subinhibitory concentrations. Allergy, 2021; 76(11):3446–3458; doi: 10.1111/all.14883

19.

Luo

, Jiang

, Jin

, et al. Exploring a phage-based real-time PCR assay for diagnosing acinetobacter baumannii bloodstream infections with high sensitivity. Anal Chim Acta, 2018; 1044:147–153; doi: 10.1016/j.aca.2018.09.038

20.

Luo

, Xie

, Liu

, et al. Bactericidal synergism between phage YC# 06 and antibiotics: a combination strategy to target multidrug-resistant Acinetobacter baumannii in vitro and in vivo. Microbiol Spectr, 2022; 10(4):e00096-22; doi: 10.1128/spectrum.00096-22

21.

, Wang

, Xu

. Studyonthecom bination of phageand antibiotics to control multi-drug resistant Acinetobacter baumannii. Chin J Antibio, 2017; 43(10):1296–1304; doi: 10.13461/j.cnki.cja.006410

22.

Macneal

, Frisbee

, Applebaum

. Therapeutic use of bacteriophages against the colon bacillus. Arch Surg, 1934; 29(2):242–247; doi: 10.1001/archsurg.1934.01180020074003

23.

Mathias

, Adam

, Simone

, et al. Enhanced antibacterial effect of the novel T4-like bacteriophage KARL-1 in combination with antibiotics against multi-drug resistant Acinetobacter baumannii. Sci Rep, 2018; 8:14140; doi: 10.1038/s41598-018-32344-y

24.

Paul

, Emily

, Kelly

, et al. Treatment strategies for persistent methicillin-resistant Staphylococcus aureus bacteraemia. J Clin Pharm Ther, 2018; 43(5):614–625; doi: 10.1111/jcpt.12743

25.

Peng

. The combined inhibitory effect of chloromycetin and bacterio phage on Salm. Typhosa. Acta microbiologica Sinica, 1959; 7(4):353–360; doi: 10.13343/j.cnki.wsxb.1959.04.006

26.

Rahman

, Kim

, et al. Characterization of induced Staphylococcus aureus bacteriophage SAP-26 and its anti-biofilm activity with rifampicin. Biofouling, 2011; 27(10):1087–1093; doi: 10.1080/08927014.2011.631169

27.

Razieh

, Susan

, Rahi

, et al. Optimization of phage-antibiotic combinations against Staphylococcus aureus Biofilms. Microbiol Spectr, 2023; 11(3):e0491822; doi: 10.1128/spectrum.04918-22

28.

Sharon

, Gavin

. Mechanisms of methicillin resistance in Staphylococcus aureus. Annu Rev Biochem, 2015; 84:577–601; doi: 10.1146/annurev-biochem-060614-034516

29.

Subramanya

, Bairy

, Metok

, et al. Detection and characterization of ESBL-producing Enterobacteriaceae from the gut of subsistence farmers, their livestock, and the surrounding environment in rural Nepal. Sci Rep, 2021; 11(1):2091; doi: 10.1038/s41598-021-81315-3

30.

Tang

, Zhang

, et al. Isolation and characterization of a broad-spectrum phage of multiple drug resistant Salmonella and its therapeutic utility in mice. Microb Pathog, 2019; 126:193–198; doi: 10.1016/j.micpath.2018.10.042

31.

Tania

, Klaus

, Hans-Peter

. Viruses versus bacteria-novel approaches to phage therapy as a tool against multidrug-resistant pathogens. J Antimicrob Chemother, 2014; 69(9):2326–2336; doi: 10.1093/jac/dku173

32.

Vanessa

, Sara

, Andreia

, et al. Staphylococcus aureus and MRSA in Livestock: Antimicrobial resistance and genetic lineages. Microorganisms, 2023; 11(1):124; doi: 10.3390/microorganisms11010124

33.

Wang

, Wu

, Qu

, et al. Isolation and characterization of a podoviridae infecting staphylococcus aureus of bovine mastitis. Chinese J Animal Infect Dis, 2022; 30(4):104–109; doi: 10.19958/j.cnki.cn31-2031/s.2022.04.028

34.

Wei

, Cong

, Li

, et al. Application of bcteriophage to prevent and control bacterial infection in livestock and poultry. China Animal Husbandry & Veterinary Med, 2020; 47(1):190–200; doi: 10.16431/j.cnki.1671-7236.2020.01.023

35.

Wernicki

, Nowaczek

, Urban-Chmiel

. Bacteriophage therapy to combat bacterial infections in poultry. Virol J, 2017; 14(1):179; doi: 10.1186/s12985-017-0849-7

36.

Xiao

, Liu

, Zhao

, et al. Prevalence and antimicrobial resistance of MSSA and MRSA isolated from different poultry. Chinese J Veterinary Drug, 2023; 57(1):1–11; doi: 10.11751/ISSN.1002-1280.2023.01.01

37.

Zeaki

, Johler

, Skandamis

, et al. Environmental parameters in staphylococcal food poisoning and resulting challenges to risk assessment. Front Microbiol, 2019; 10(10):1307; doi: 10.3389/fmicb.2019.01307

38.

Zhang

, Yan

, Yang

, et al. Rapid and selective detection of E. coli O157: H7 combining phagomagnetic separation with enzymatic colorimetry. Food Chem, 2017; 234:332–338; doi: 10.1016/j.foodchem.2017.05.013

39.

Zhao

, Zhao

, Zhai

, et al. Biological characterization and genomic analysis of a novel methicillin-resistant Staphylococcus aureus phage, SauPS-28. Microbiol Spectr, 2024; 12(2):e0029523–e17; doi: 10.1128/spectrum.00295-23