The Phage Holin HolGH15 Exhibits Potential As an Antibacterial Agent to Control Listeria monocytogenes

Abstract

Listeria monocytogenes is an important foodborne pathogen that is a serious threat to public health security, and new strategies to control this bacterium in food are needed. HolGH15, derived from Staphylococcus aureus phage GH15, has shown antibacterial activity against several bacterial species. In this work, the antilisterial behavior and effectiveness of HolGH15 are further studied. To elucidate its antimicrobial modes against L. monocytogenes, cell integrity and membrane permeabilization assays were performed. When treated with HolGH15, the release of 260-nm-absorbing materials of L. monocytogenes was rapidly increased. HolGH15 triggered a significant increase in fluorescence intensity by flow cytometry. In membrane permeabilization assays, the cytoplasmic β-galactosidase of L. monocytogenes treated with HolGH15 was released via an increase in the permeability of the membrane. HolGH15 caused changes in the structural properties of L. monocytogenes cells resulting in shrinkage, which evoked the release and removal of cellular contents and finally lead to cell death. Electron microscopy observations indicated that HolGH15 exhibited excellent bactericidal potency by permeabilizing the cell membrane, damaging membrane integrity, and inducing cellular content shrinkage or loss. Moreover, HolGH15 (at the final concentration of 240 μg/mL) reduced L. monocytogenes (at the initial concentration of 10⁶ colony-forming unit/mL) to an undetectable level at 4°C. Collectively, HolGH15 has potential as a novel antimicrobial agent against L. monocytogenes in the manufacture and store of food by spraying or soaking, especially at refrigerated temperature.

Introduction

L isteria monocytogenes is an important foodborne pathogen that is a serious threat to public health security (Schlech, 2000; Borcan et al., 2014; Lomonaco et al., 2015). L. monocytogenes is widely distributed in the environment, and can grow at refrigerated temperatures and is highly tolerant of acidity and salinity (McLauchlin et al., 2004; Bergholz et al., 2018). These characteristics make L. monocytogenes difficult to control (de Castro et al., 2012; Almeida et al., 2013). New strategies to control L. monocytogenes are therefore urgently needed.

One such strategy is to use the lytic activities of bacteriophage (phage), which offer an effective and environmentally friendly method to reduce bacterial contamination (Oliveira et al., 2014; Figueiredo and Almeida, 2017; Ibarra-Sanchez et al., 2018). However, phages are bacterial virus, and their genomes may carry genes related to lysogenic, bacterial drug resistance and virulence, which have certain safety risks (Copin et al., 2019). Holin is encoded by phage and has been proven to effectively kill Gram-positive bacteria from the extracellular, including Streptococcus suis and Staphylococcus aureus (Shi et al., 2012a; Song et al., 2016). By contrast, holin is a protein that is safer and more effective than bacteriophage to control L. monocytogenes during the processing and preservation of food (such as meat products, eggs, fruits, and vegetables) by spraying or soaking (Chang, 2020).

Holins have been identified in a wide variety of phages (Wang et al., 2000; Donovan, 2007; Catalao et al., 2010). Holins possess certain common characteristics (Shi et al., 2012b). For example, holins at least contain one transmembrane α-helical sequence that is essential for function and consist of a highly charged and hydrophilic C-terminal domain (Lella et al., 2016). Most holins are encoded by a gene adjacent to the endolysin gene (Reddy and Saier, 2013). In the late period of phage infecting the host bacteria, phage-encoded holin proteins can accumulate in the cytoplasmic membrane before triggering; when the concentration of holin proteins reaches a threshold, micron-scale holes will be formed, which allow the soluble endolysin to release from the cytoplasm to reach the peptidoglycan in the cell wall (Young, 2014; Fernandes and São-José, 2016).

HolGH15, the holin of the staphylococcal phage GH15, consists of 167 amino acids and belongs to the phage_holin_1 superfamily (Gu et al., 2013; Song et al., 2016). Previous studies have shown that HolGH15 has a broad antibacterial range, not only does it inhibit S. aureus but also other species, including L. monocytogenes (Song et al., 2016). However, the mechanism of action of HolGH15 has not been subjected to systematic in-depth research. Therefore, the aim of this study is to investigate the antimicrobial activity and elucidate the antimicrobial mechanism of HolGH15 against L. monocytogenes.

Materials and Methods

HolGH15, strains, and culture conditions

HolGH15 was prepared as described previously (Song et al., 2016). The Escherichia coli BL21-CodonPlus cells (Stratagene), which contains pET-28a-HolGH15, were cultured and induced with isopropyl β-D-thiogalactoside (IPTG) at 25°C and 180 r/min. HolGH15 was purified using HisPur Ni-NTA Resin (Pierce Biotechnology). L. monocytogenes strains, American Type Culture Collection (ATCC) 19112 (serotype 2a), ATCC 19115 (serotype 4b), and ATCC 15313 (serotype 1/2a), were purchased from the ATCC. L. monocytogenes was cultured at 37°C for 4 h and grown to exponential phase (optical density [OD]₆₀₀ = 0.5) in the brain/heart infusion (BHI) broth (BD) with rotary shaking at 180 r/min before use. The strains were stored at −80°C in 25% glycerol. Polymyxin acriflavine lithium chloride ceftazidime aesculin mannitol (PALCAM) agar was used for L. monocytogenes isolation.

Measurement of antimicrobial activity

To determine the antibacterial activity of HolGH15, different serotypes of L. monocytogenes were used as an indicator strain. HolGH15 (at the final concentration of 300 μg/mL) was spotted onto freshly seeded lawns of L. monocytogenes, and the production of transparent antibacterial ring was assessed after incubation for 12 h at 37°C. Bacterial cells were washed and resuspended with sterile phosphate-buffered saline (PBS) to 5 × 10⁵ colony-forming unit (CFU)/mL. HolGH15 (at the final concentration of 300 μg/mL) was added to the bacterial suspension, and the mixture was incubated for 60 min at 37°C. The antibacterial activity was expressed as the CFU reduction following exposure. As a negative control, the bacterial strains were treated with elution buffer (PBS with 150 mM imidazole; pH 7.4) under the same conditions.

Minimum bactericidal concentration (MBC) assays

A bacterial growth inhibition assay of HolGH15 against L. monocytogenes was conducted using a standard microdilution method as recommended by the Clinical and Laboratory Standards Institute (NCCLS, 2002). Fresh cultures of the tested strains were inoculated into 96-well plates (Corning) at a final inoculum of 10⁵ CFU/mL. Serial twofold dilutions of HolGH15 with PBS were performed. After a 24-h incubation at 37°C, the presence or absence of growth was observed in each well. The minimal inhibitory concentration (MIC) has been observed in our previous study (Song et al., 2016). The MBC is determined by reculturing (subculturing) broth dilutions that inhibit growth of a bacterial organism (i.e., those at or above the dose of the MIC). The broth dilutions are streaked onto agar and incubated for 24–48 h and colonies were enumerated to determine viable CFU/mL. The MBC was the lowest concentration that demonstrated a predetermined reduction (e.g., 99.9%) in CFU/mL. The broth containing no microbial cells was used as the negative control, and each test was repeated three times using six replicates.

Bacterial cell integrity assay

The integrity of bacterial cell membrane was examined by determining the release of material (including DNA, RNA, and macromolecules) absorbing at 260 nm (Chen and Cooper, 2002). Bacterial cultures were harvested by centrifugation at 5000 × g for 10 min, washed, and resuspended in 0.5% NaCl solution. The final cell suspension was adjusted to an absorbance of 0.5 at 600 nm. Then, 2 mL of HolGH15 (240 μg/mL) was mixed with 2 mL of bacterial cell suspension, and the release of materials absorbing at 260 nm was monitored over time (0, 20, 40, 60, 80, 100, and 120 min) with an ultraviolet spectrometer. L. monocytogenes ATCC 19112, which has not been treated with HolGH15, was a control.

Flow cytometry

The damage of cellular membrane was determined by flow cytometry. L. monocytogenes ATCC 19112 (ca. 10⁵ CFU/mL) was mixed with HolGH15 (240 μg/mL) at 37°C for 30 min. L. monocytogenes ATCC 19112, which has not been added to HolGH15, was a control. Then, a final concentration of 10 μg/mL of propidium iodide (PI) was added to the bacterial suspension and incubated for 30 min. The bacterial cells were harvested and resuspended in PBS. Flow cytometry was performed using a FACScan (Becton-Dickinson, San Jose, CA).

Permeabilization assay

The cytoplasmic membrane permeabilization of L. monocytogenes ATCC 19112 treated with HolGH15 was assessed by measurement of β-galactosidase activity using O-nitrophenyl-beta-D-galactopyranoside (ONPG), which is a substrate for cytoplasmic β-galactosidase enzyme (Zhu et al., 2014). In brief, L. monocytogenes ATCC 19112 at the midlog phase was adjusted to 10⁵ CFU/mL with 10 mM PBS (pH 7.4, containing 1.5 mM ONPG), and then treated with HolGH15 (at the final concentration of 240 μg/mL) for 4 h at 37°C. Hereafter, Na₂CO₃ (0.5 mol/L) was added into the mixture to stop the reaction and then the mixture was centrifuged at 8000 × g for 10 min. The OD₄₂₀ of the supernatant was detected. β-galactosidase activity (U/mL) = (OD₄₂₀ × V)/(T × VS × 0.0045). V: the volume of the mixture (mL); T: the time of reaction (min); VS: the volume of sample that was used to detect OD₄₂₀ (mL); 0.0045: the extinction coefficient (mL/nmol). The same method was used to determine the cytoplasmic membrane permeabilization of E. coli BL21-CodonPlus cells containing pET-28a or pET-HolGH15 induced by IPTG. E. coli BL21-CodonPlus cells uninduced by IPTG were used as a control.

Microscopy

L. monocytogenes ATCC 19112 was cultured at midlog phase, and the bacteria were washed three times and finally resuspended using PBS. L. monocytogenes was diluted to 10⁶ CFU/mL. Briefly, 100 μL of L. monocytogenes and 100 μL of HolGH15 (at a final concentration of 120 μg/mL) were added into the BHI culture medium and incubated at 4°C for 4 h. Samples were Gram stained and observed by optical microscopy.

Morphological changes in L. monocytogenes cells were visualized after exposure to HolGH15 (120 μg/mL) for 4 h at 4°C by scanning electron microscopy (SEM; XL30ESEM-FEG; FEI). Ultrastructural changes in L. monocytogenes cells were visualized after exposure to HolGH15 (120 μg/mL) for 4 h at 4°C by transmission electron microscopy (TEM; H-7650; Hitachi). The samples were prepared and fixed as described previously (Ma et al., 2015).

The antibacterial activity of HolGH15 on L. monocytogenes at refrigerated temperatures

The antibacterial activity of HolGH15 on L. monocytogenes was tested at 4°C. L. monocytogenes ATCC 19112 was cultured and harvested at midlog phase, and the bacteria were washed three times and finally resuspended using PBS (pH 7.2). L. monocytogenes was diluted to 10⁶ CFU/mL. Briefly, 100 μL of L. monocytogenes and 100 μL of HolGH15 (at a final concentration of 120 or 240 μg/mL) were added to the BHI culture medium and incubated at 4°C. Samples were collected each day for a week. Remaining L. monocytogenes cells were enumerated by colony counts on PALCAM agar plates (Hopebio, Qingdao, China). The experiments were repeated in triplicate.

Statistical analysis

t-Test analysis of variance was performed by using the GraphPad Prism 5 software package (GraphPad Software, Inc., San Diego, CA). The data are presented as mean ± standard deviation from at least three independent experiments. The statistical significance was defined as a p-value of <0.05.

Results

Antimicrobial activity of HolGH15

The antimicrobial abilities of HolGH15 against L. monocytogenes are shown in Figure 1. Clear rings were observed when HolGH15 was spotted onto BHI agar plates covered with a bacterial lawn of L. monocytogenes. Log-phase cultures of different serotypes of L. monocytogenes were exposed to HolGH15 (final concentration, 300 μg/mL) for 60 min. The results revealed a reduction of three serotype L. monocytogenes (5.27 log₁₀ CFU/mL reduction of ATCC 19112, 5.37 log₁₀ CFU/mL reduction of ATCC 19115, and 4.97 log₁₀ CFU/mL reduction of ATCC 15313) after exposure to HolGH15. Reductions of controls were very less (0.43 log₁₀ CFU/mL reduction of ATCC 19112, 0.39 log₁₀ CFU/mL reduction of ATCC 19115, and 0.23 log₁₀ CFU/mL reduction of ATCC 15313). The MBC values for HolGH15 against different serotypes of L. monocytogenes are 240 μg/mL. The results indicated that different serotypes of L. monocytogenes exhibited similar sensitivity to HolGH15.

FIG. 1.

The antibacterial activity of HolGH15. (A) Plate lytic assay. The antibacterial activity of HolGH15 (300 μg/mL) against Listeria monocytogenes was tested using plate assays. (B) Antibacterial activity of HolGH15 against L. monocytogenes. Different serotypes of L. monocytogenes (5 × 10⁵ CFU/mL) were exposed to HolGH15 (300 μg/mL) for 60 min at 37°C. Antibacterial activity in vitro was expressed as the CFU reduction. L. monocytogenes, which has not been treated with HolGH15, was used as control. The values represent the mean ± SD (n = 3). CFU, colony-forming unit; SD, standard deviation.

Cell integrity

In considering the mechanism of action used by holin, we hypothesized that HolGH15 might induce bacterial membrane damage. The release of intracellular components from L. monocytogenes ATCC 19112 suspensions treated with HolGH15 is shown in Figure 2. The absorbance at 260 nm was increased in a time-dependent manner upon the addition of HolGH15, and the release of intracellular components following treatment with HolGH15 was higher than that of the control group. Up to 20 min, L. monocytogenes treated with HolGH15 (OD₂₆₀ = 0.61) was significantly higher than the control group (OD₂₆₀ = 0.21); thereafter, the absorbance gradually increased and generally stabilized.

FIG. 2.

The dynamic antibacterial activity of HolGH15 against Listeria monocytogenes. L. monocytogenes ATCC 19112 was treated with 240 μg/mL of HolGH15 (black circles). L. monocytogenes ATCC 19112 without HolGH15 as a control (white circles). Every line with black or white circles represents the release of L. monocytogenes extracellular materials absorbing at 260 nm treated with HolGH15 or not. The values represent the mean ± SD (n = 3). ATCC, American Type Culture Collection; SD, standard deviation.

PI fluorescently stained the nucleic acids of cells when the cells suffered disruption of the cytoplasmic membrane integrity. Compared with the fluorescence released in the case of no treatment, the addition of HolGH15 was significantly increased by 83.4% in fluorescence intensity (Fig. 3), indicating strong membrane integrity damage.

FIG. 3.

The damage of cellular membrane determined by flow cytometry. (A) Flow cytometry analysis of Listeria monocytogenes. The increments of cellular fluorescence intensity of PI (10 μg/mL) after a 30-min treatment with HolGH15 at 1 × MIC were monitored by flow cytometry. (B) The percentage of PI was absorbed by L. monocytogenes ATCC 19112. ATCC, American Type Culture Collection; MIC, minimal inhibitory concentration; PI, propidium iodide.

Cell membrane permeabilization of L. monocytogenes

The permeabilization of L. monocytogenes was evaluated as a function of cytoplasmic β-galactosidase release. As shown in Figure 4, the release of cytoplasmic β-galactosidase was significantly increased (0.81 U/mL) following treatment with HolGH15 in L. monocytogenes suspensions. Similarly, the release of cytoplasmic β-galactosidase activity (0.61 U/mL) in E. coli BL21-CodonPlus cells induced by HolGH15 was obviously higher than those (0.25 U/mL) uninduced by HolGH15 (p < 0.01). This result also supported that HolGH15 has cell membrane permeabilization activity.

FIG. 4.

Determination of extracellular β-galactosidase activity. The release of cytoplasmic β-galactosidase was detected. Listeria monocytogenes ATCC 19112 was treated with HolGH15 (120 μg/mL) for 4 h. Escherichia coli BL21-CodonPlus cells contains pET-28a and pET-HolGH15 induced by IPTG for 4 h. E. coli BL21-CodonPlus cells uninduced as control. The values represent the mean ± SD (n = 3). ATCC, American Type Culture Collection; IPTG, isopropyl β-D-thiogalactoside; SD, standard deviation.

Morphological and ultrastructural properties of L. monocytogenes treated with HolGH15

Gram staining is a widely used microbiological staining technique that greatly aids in the identification and characterization of bacteria. The Gram reaction reflects fundamental differences in the biochemical and structural properties of bacteria. As shown in Figure 5, a part of counterstain L. monocytogenes cells were red, as observed by optical microscopy, and changes in the structural properties of L. monocytogenes were observed after incubation with HolGH15.

FIG. 5.

Morphological and ultrastructural images of Listeria monocytogenes treated by HolGH15 at refrigerated temperature (4°C). L. monocytogenes ATCC 19112 cells were exposed to HolGH15 and observed by optical microscopy (A, B), SEM (C, D) and TEM (E, F), respectively. (A, C, E) Represent untreated cells (control), while (B, D, F) show cells treated with HolGH15 at 120 μg/mL. ATCC, American Type Culture Collection; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

SEM and TEM images of L. monocytogenes cells exposed to HolGH15 showed ultrastructural and morphological changes (Fig. 5). Compared with untreated L. monocytogenes cells, bacterial cells exposed to HolGH15 exhibited remarkable cellular damage, resulting in obvious cell shrinkage and rupture. At the MIC, HolGH15 provoked the rough texture and shrinkage of L. monocytogenes, according to our SEM observations. The cellular contents of L. monocytogenes became almost undetectable, which were visible by TEM. In addition, cell membranes and cell walls were disrupted and damaged, resulting in the release or condensation of cellular contents (Fig. 5).

The antibacterial activity of HolGH15 against L. monocytogenes at 4°C

The ability of HolGH15 to inhibit L. monocytogenes ATCC 19112 was tested at 4°C. At a concentration of 240 μg/mL (2 × MIC), the number of viable L. monocytogenes ATCC 19112 was reduced to undetectable levels after 5 days (Fig. 6). This result represented a reduction of greater than 5 log₁₀ CFU/mL compared with the control group. At a concentration of 120 μg/mL (MIC), viable counts were reduced by ∼4.03 log₁₀ CFU/mL. Thus, HolGH15 (240 μg/mL) has the ability to reliably control L. monocytogenes at 4°C.

FIG. 6.

The antibacterial activity of HolGH15 against Listeria monocytogenes at refrigerated temperature (4°C). L. monocytogenes ATCC 19112 (10⁶ CFU/mL) were exposed to HolGH15 (1 × MIC and 2 × MIC) for 7 days at 4°C. At the indicated times, bacterial counts (CFU/mL) treated with 120 μg/mL of HolGH15 (black squares), 240 μg/mL of HolGH15 (black circles) or PBS (black triangles) were determined by colony count. The values represent the mean ± SD (n = 3). ATCC, American Type Culture Collection; CFU, colony-forming unit; MIC, minimal inhibitory concentration; PBS, phosphate-buffered saline; SD, standard deviation.

Discussion

HolGH15 derived from S. aureus bacteriophage GH15 showed a broad antibacterial range (Song et al., 2016). In particular, higher susceptibility to HolGH15 was observed in L. monocytogenes (Song et al., 2016). Based on the results of testing the antibacterial spectrum of HolGH15, L. monocytogenes strains from different serotypes (2a, 4b, 1/2a) were sensitive to HolGH15. In addition, the MBC of HolGH15 against different serotypes L. monocytogenes was identical. HolGH15 showed bactericidal effects on L. monocytogenes, whereas it exerted bacteriostatic activity on S. aureus (Song et al., 2016).

To gain insight into the molecular mechanism of action of HolGH15, we performed a series of molecular and cellular assays on the bacterial membrane. The cytoplasmic cell membrane is commonly the target for many antimicrobial agents (Je and Kim, 2006). When antimicrobial agents interact with bacterial membranes, membrane changes are induced that are initially indicated by the leaching out of low-molecular-mass species (Chen and Cooper, 2002). The absorbance at 260 nm was dramatically increased in a time-dependent manner upon the addition of HolGH15. This result indicated that HolGH15 may disrupt the cell membrane integrity, causing release of DNA, RNA, and macromolecules. To further investigate cytoplasmic membrane integrity, flow cytometry was used to analyze the cells by staining with PI. The addition of HolGH15 triggered a significant increase in fluorescence intensity, further suggesting that HolGH15 possessed the ability to damage the bacterial cell membrane. Cell membrane permeabilization of L. monocytogenes was evaluated as a function of cytoplasmic β-galactosidase release. Normally, β-galactosidase is present inside the cell and is not released outside the cell. The release of cytoplasmic β-galactosidase was significantly increased following treatment with HolGH15 in L. monocytogenes suspensions. This confirmed the cell membrane permeabilization activity of HolGH15, and verified the results of the cell integrity assay.

The morphological alterations in L. monocytogenes were further analyzed by microscopy. First, Gram staining was used to illustrate fundamental differences in the biochemical and structural properties of L. monocytogenes. After treatment with HolGH15, some L. monocytogenes became red after Gram staining. It is possible that HolGH15 caused the damage of the integrity of peptidoglycan (Obiang-Obounou et al., 2011), during the process of passing through the peptidoglycan. Then, more direct visual observations by SEM and TEM further confirmed the significant membrane damage caused by HolGH15. Owing to the depolarization and destruction of membrane integrity or even the micron-scale holes caused by HolGH15, L. monocytogenes became shrinkage and rough texture under the differential osmotic pressures inside and outside of the cell. These results confirmed that the target activity allows HolGH15 to cause membrane permeabilization, which is followed by disruption of the cytoplasmic membrane, leading to leakage, the lethal event leading to bacterial cell death. This mechanism of membrane disruption makes HolGH15 an attractive candidate for the development of promising therapeutic/antibacterial agents against L. monocytogenes.

L. monocytogenes is an opportunistic foodborne pathogen responsible for listeriosis, a disease associated with high mortality rates (up to 30%) (Lomonaco et al., 2015). The optimal growth conditions for L. monocytogenes are refrigerated temperatures and high acidity. L. monocytogenes is also salt-tolerant; all of these characteristics contribute to the difficulties in controlling this pathogen in food (McLauchlin et al., 2004). Based on the optimal activity range conditions for HolGH15, it has the potential to be effective in the control of L. monocytogenes. The antimicrobial effects exerted by HolGH15 on L. monocytogenes in BHI at refrigeration temperatures (4°C) have been evaluated. In the absence of antimicrobial treatment, L. monocytogenes growth was observable. HolGH15 at 120 μg/mL was able to inhibit L. monocytogenes growth within 7 days. When HolGH15 was used at a concentration of 240 μg/mL, the concentration of L. monocytogenes reduced by 5 log₁₀ CFU/mL. HolGH15 decreased the viable counts of L. monocytogenes ATCC 19112 to undetectable levels within 5 days at 4°C. HolGH15 showed effective bactericidal and bacteriostatic activities against L. monocytogenes at refrigeration temperatures. In our previous study, HolGH15 has no hemolytic activity and is relatively safe for human. HolGH15 has different degrees of antibacterial activity at a pH between 4 and 8.5. It has the potential to control L. monocytogenes in fruit slices and juices (Oliveira et al., 2014). HolGH15 is expected to be able to control L. monocytogenes during food preservation and meat processing. For example, meat products, fruits, and vegetables can be directly sprayed or soaked with HolGH15 as a means of preservation. Thus, the application of HolGH15 represents a safe, environmentally friendly, promising, and chemical-free alternative to control L. monocytogenes.

Conclusions

In this study, we elucidated the antimicrobial mechanism of action of HolGH15 and confirmed the antibacterial capability of L. monocytogenes at refrigerated temperatures. Its ability for membrane disruption makes HolGH15 an attractive candidate for the development of promising therapeutic/antibacterial agents against L. monocytogenes. During the processing and preservation, meat products, eggs, fruits, and vegetables can be directly sprayed or soaked with HolGH15.

Footnotes

Disclosure Statement

We confirm that all the authors have fulfilled the conditions required for authorship. There is no potential conflict of interest or financial dependence pertaining to this publication, as described in the Instructions for Authors. All authors have read and approved the article.

Funding Information

This study was supported by the National Natural Science Foundation of China (project Nos. U19A2038, 31872505, and 31802226), the Natural Science Foundation of Jilin Province (Changchun, China; grant No. 20200201120JC), the Jilin Province Science Foundation for Youths (Changchun, China; grant No. 20190103106JH), the Natural Science Foundation of Heilongjiang Province of China (QC2017021), the Heilongjiang Postdoctoral Science Foundation Grant (LBH-Z17185), the Postdoctoral Science Foundation of Heilongjiang Bayi Agricultural University, and the Fundamental Research Funds for the Central Universities.

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