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Isolation and relationship analysis of Listeria phages with various serotype hosts and morphological characterization

Virology Journal volume 22, Article number: 104 (2025) Cite this article

Abstract

Listeriosis, caused by Listeria monocytogenes (Lm), is a severe foodborne illness with a high fatality rate. Listeria phages specifically target and lyse Lm, offer a promising alternative for biocontrol and phage therapy. However, most existing studies focus on the lytic characteristics of Listeria phages using limited sample sizes. In this study, a large number of Listeria phages were isolated from diverse sources, and their lytic profiles and morphology were characterized. A total of 317 Listeria phages were isolated from 90 food-related environmental samples and 196 natural environmental samples collected across seven provinces. The phages were tested for lytic activity against 35 Lm strains representing nine serotypes, and their morphology was characterized using transmission electron microscopy (TEM). Statistical analysis was conducted to evaluate the lytic patterns of phages. The phages were classified into three groups based on their total lysis ratios. Broad Host Range Phages (BHRP) were primarily members of the Myoviridae-like phages and demonstrated the ability to lyse a vast majority of nine serotype host strains. Medium Host Range Phages (MHRP) comprised both Siphoviridae-like and Myoviridae-like phages, and demonstrated lysis of 6–9 serotype strains. Narrow Host Range Phages (NHRP) belonged to the Siphoviridae-like phages and exhibited effective lysis of serotype 4 strains. Furthermore, phages isolated from food-related environmental sources demonstrated greater lytic activity against Listeria serotypes 1/2b, 4a, and 4c compared to those derived from natural environmental sources. The study first isolated a multitude of Listeria phages, elucidated their lytic patterns and ecological distribution, and provided a valuable resource for future research.

Introduction

Listeriosis is the third most significant cause of mortality from foodborne pathogens, with a fatality rate of 20 to 30% [1]. Listeria monocytogenes, a ubiquitous pathogen responsible for listeriosis, has the capacity to survive and proliferate over a broad range of temperatures (2–45\(^\circ \,\hbox {C}\)) and pH levels (4.6\(-\)9.5), as well as in the elevated salt concentrations [2, 3]. L. monocytogenes, is a genetically heterogeneous species comprising 14 serotypes (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4ab, 4c, 4d, 4e, 4 h and 7) that can be grouped into five distinct serogroups using a multiplex PCR scheme: IIa (1/2a, 3a), IIb (1/2b, 3b, 7), IIc (1/2c, 3c), IVb (4b, 4d, 4e) and L (4a, 4c, 4ab) [4,5,6]. Epidemiological studies have demonstrated that the 1/2a, 1/2b, 1/2c, 4b serotype strains are prevalent in food contamination [7, 8], and the 4b, 1/2a, and 1/2b serotype strains are responsible for 95% of human listeriosis cases [9].

Bacteriophages (phages) are viruses that are capable of specifically infecting and killing bacteria. Lytic phages infect and kill their bacterial host through a process known as lysis. The interest in bacteriophage therapy is growing, which can be attributed to the exceptional lysis specificity of these viruses [10,11,12,13,14].Phages host range refers to the spectrum of bacterial strains a phage can infect. Broad host range phages can lyse diverse strains across multiple serotypes, whereas narrow host range phages are restricted to a few closely related strains. However, no standardised definition distinguishes broad and narrow host range phages [15]. Listeria-specific bacteriophages, also referred to as Listeria phages, have been isolated from various sources. To date, more than 500 Listeria phages have been identified. Nevertheless, only a limited number of virulent bacteriophages with the potential for Listeria biocontrol have been fully characterized at the molecular and genomic level [8, 9, 16,17,18]. Although several studies have been conducted on the lysis characteristics of Listeria phages, the sample size was relatively limited, and the analysis was not sufficiently systematic [16, 17, 19].

This study aimed to isolate a diverse range of Listeria phages, examine their relationships with various serotype host strains, and investigate their morphological characteristics.

Methods

Sample collection

A total of 90 samples from food-related environments and 196 samples from natural environments were collected from seven provinces. The 90 food-related environmental samples comprise 58 seafood samples (obtained from the seafood markets), 7 swab samples (taken from the surface of pork cutting boards), 3 frozen food samples (frozen meet wontons, frozen hotpot meatballs and frozen dumplings), and 22 market sewage samples. The 196 natural environmental samples include 63 soil samples, 88 sand samples, 2 spring water samples, and 43 seawater samples (Table 1).

Table 1 Samples and isolated phages in this study
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Listeria monocytogenes strains

A total of 31 strains (referred to as isolation strains), including 21 laboratory-preserved strains and 10 newly isolated from the samples, were employed to isolate phages (Table 2). A total of 35 strains (referred to as host strains) were employed to assess the lytic activity of the isolated phages. The isolation strains and host strains are represented by nine serotypes (1/2a, 1/2b, 1/2c, 4b, 4d, 4a, 4c, 3a, 3b) of L. monocytogenes, respectively (Table 2).

Table 2 Isolation strains and host strains used in this study
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Isolation of phages

In accordance with the methodology outlined in reference [19,20,21], the samples were subjected to an overnight enrichment process in BHI broth, supplemented with 31 distinct isolation strains and CaCl2. Subsequently, the cultures were collected and filtered. A portion of each filtered culture was combined with an individual isolated strain at a specified optical density (OD). Then the mixtures were incubated for a period of 24 to 48 h. Subsequently, the double-layer agar method applied bacteriophage mixtures to solid agar. The identification of the phages was conducted by observing plaque formation, with the plaques subsequently undergoing purification on three occasions and stored at 4 °C.

Host-range test

The lytic activity of bacteriophages was evaluated by measuring optical density at 600 nm (OD600). In brief, overnight cultures of host bacterial strains were initially diluted 1:100 in BHI medium and incubated at 37 \(^\circ\hbox {C}\) with shaking at 200 rpm for 2 h. Subsequently, 100 \(\mu\)L of each bacterial suspension was transferred into individual wells of a 96-well cell culture plate (Corning). Subsequently, 100 \(\mu\)L of either the phage suspension or BHI broth (which served as the negative control) was added to each well and the plate were incubated for 2-3 h at 30\(^\circ\hbox {C}\). The OD600 for each well was determined using a microplate reader (BioTek, Washington, DC, USA). The C value is calculated as follows: C value = (OD600 of negative control wells - OD600 of test wells) / OD600 of negative control wells. A C value exceeding 0.22 was deemed indicative of positive lysis.

Serotype-specific lysis ratio calculation

The serotype-specific lysis ratio was calculated as the proportion of host strains within a given serotype that were lysed by a phage. This was expressed as follows: Serotype-specific lysis ratio = (number of serotype-specific lysis-positive host strains/ total number of tested serotype-specific host strains) ∗ 100%. This ratio was used to assess the efficiency of phage lysis across different serotypes.

Transmission electron microscopy

The purified phages were adsorbed onto formvar and carbon film-coated grids for one minute, and then stained with 1% (w/v) phosphotungstic acid (pH 6.8) for one minute. Following air drying, the grids were observed using a Tecnai 12 transmission electron microscope (FEI, Eindhoven, Netherlands) at 120 kV, with images captured using a charge-coupled device (CCD) camera.

Statistical analysis

The Wilcoxon rank-sum test was utilized to analyze the distribution of binary categorical variables. All statistical analyses were conducted using the R statistical computing software (version 4.4.0).

Results

Phages isolation of samples

A total of 317 Listeria phages were isolated, comprising 57 from 90 food-related environmental samples and 260 from 196 natural environmental samples (Table 1). Phages with identical lytic profiles from the same samples were excluded from further analysis, leaving 293 distinct phages for consideration.

Relationship between Listeria phages and host strains

In order to elucidate the ecological roles of phages, the phages were categorized into three groups based on total lysis ratios (total lysis ratio = lysis positive host strains / all tested host strains) against host strains: Broad Host Range Phages (BHRP) with a lysis ratio>0.85; Medium Host Range Phages (MHRP), with a lysis ratio between 0.38 and 0.85; and Narrow Host Range Phages (NHRP), with a lysis ratio<0.38. Of the 293 phages, 33 were identified as BHRP, 204 as MHRP, and 56 as NHRP.

Susceptibility of various serotype strains to phage

Broad Host Range phages (BHRP) Among the broad host range phages (BHRP), all nine tested serotype strains (including those frequently associated with food contamination and human listeriosis) were susceptible to the phages. However, a few individual strains, such as Lm331 (4b) and Lm258 (1/2c), exhibited resistance (Fig. 1; Tables 4, 5, 6).

Fig. 1
figure 1

Lysis Profiles of Broad Host Range Phages (BHRP) (n = 33). Left labels indicate phage sources and sample types, while right labels denote provinces of origin. Columns represent host strains, annotated with their serotypes

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Medium Host Range Phages (MHRP) Among the medium host range phages (MHRP), the susceptibility of the nine serotype strains to phages was found to vary. For serotypes frequently associated with food contamination (1/2a, 1/2b, 1/2c, 4b) and human listeriosis (1/2a, 1/2b, 4b), serotype 4b was found to be the most susceptible to phages, while serotype 1/2b was found to be the least susceptible. Notably, 1/2b strains exhibited low susceptibility to phages derived from natural environmental sources yet demonstrated comparable susceptibility to those derived from food-related environmental sources, similar to that observed in 1/2a and 1/2c strains (Table 3; Fig. 2). A total of 80.39% of the phages tested were found to be effective against strains belonging to 8 or 9 serotypes, as indicated by the number of phages lysing these serotypes divided by the total number of phages in the MHRP (Table 4). The data further indicated that 78.92% of MHRP was effective against strains from all serotypes associated with food contamination (Table 5), and 80.39% were effective against all serotype strains associated with human listeriosis (Table 6). These results were based on the phages lysing at least one strain of each serotype. Moreover, strains from nine serotypes exhibited high susceptibility to MHRP, showing 53.85% susceptibility to phages isolated from food-related environmental sources and 52.12% susceptibility to phages derived from natural environmental sources (Table 4).

Table 3 Sensitivity of various serotypes strains to different phage groups
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Fig. 2
figure 2

Lysis Profiles of Medium Host Range Phages (MHRP) (n = 204). Left labels indicate phage sources and sample types, while right labels denote provinces of origin. Columns represent host strains, annotated with their serotypes

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Table 4 Number of serotypes lysed by various phage groups
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Table 5 Number of serotype commonly found in food contamination (1/2a, 1/2b, 1/2c, 4b) lysed by various phage groups
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Table 6 Number of serotype commonly found in human listeriosis cases (1/2a, 1/2b, 4b) lysed by various phage groups
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Narrow Host Range Phages (NHRP) It was demonstrated that strains of serotype 4a and 4b exhibited high susceptibility to the NHRP, while strains of other serotypes exhibited relatively low susceptibility (Fig. 3; Table 3). Furthermore, strains from five to seven serotypes demonstrated susceptibility to 80.36% of the phages, calculated as the ratio of phages lysing five to seven serotypes to the total number of phages in the NHRP (see Table 4 for details). Additionally, 21.43% of the phages were effective against all serotype strains associated with food contamination (Table 5), and 26.79% were effective against all serotype strains associated with human listeriosis (Table 6). Furthermore, strains from five to six serotypes were susceptible to 75.00%(calculated as the ratio of phages lysing five and six serotypes to total phages sourced from the food-related environment in the NHRP) of phages derived from food-related environmental sources, while strains from 6 to 7 serotypes exhibited susceptibility to 56.25% (calculated as the ratio of phages lysing 6 and 7 serotypes to total phages sourced from the natural environment in the NHRP) of phages derived from natural environmental sources (Table 4).

Fig. 3
figure 3

Lysis Profiles of Narrow Host Range Phages (NHRP) (n = 56). Left labels indicate phage sources and sample types, while right labels denote provinces of origin. Columns represent host strains, annotated with their serotypes

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Phage lysis against different serotype strains

A comparative analysis of phages derived from food-related and natural environmental sources revealed discrepancies in serotype-specific lysis ratios.

Among the 33 broad host range phages (BHRP), the lysis ratios for serotypes were generally high, with all exceeding 90%. However, the lysis ratio for serotype 1/2c strains was relatively low (83.20%) for phages derived from natural environmental sources, and the lysis ratio for serotype 4c strains was also low (79.17%) for phages derived from food-related environmental sources (Fig. 4).

Fig. 4
figure 4

Comparison of the phage lysis activities by sample environmental sources. A Broad Host Range Phages (BHRP) (n = 33). B Medium Host Range Phages (MHRP) (n = 204). C Narrow Host Range Phages (NHRP) (n = 56). The bars represent the mean + SD. FE, Food-related environment; NE, Natural environment. Statistical analyses were conducted using the Wilcoxon rank-sum test, with the following significance levels: *P<0.05; ** P < 0.01 and ***P < 0.001

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Among the 204 medium host range phages (MHRP), the lysis ratio for serotype 4a and 4c strains were relatively high (83.76% and 88.03% for food-related environmental sources; 74.55% and 76.36% for natural environmental sources), In contrast, the lysis ratios for serotype 1/2b and 3b strains were notably lower (53.33% and 39.32% for food-related environmental sources; 39.39% and 44.24% for natural environmental sources) (Fig. 4). Notably, the lysis ratios for serotype 1/2b, 4a, and 4c strains were significantly higher for phages derived from food-related environmental sources compared to those from natural environmental sources (53.33% vs. 39.39%, P = 0.011; 83.76% vs. 74.55%, P = 0.045; and 88.03% vs. 76.36%, P < 0.001; respectively).

Among the 56 narrow host range phages (NHRP), the serotype-specific lysis ratios were generally low, except for serotype 4 strains (4a, 4b, 4c, 4d), which exhibited higher lysis ratios (66.67%, 45.00%, 58.33%, and 41.67% for food-related environmental sources; 72.22%, 47.08%, 79.86%, and 33.33% for natural environmental sources) (Fig. 4). Notably, no phage was found to exclusively target a single serotype.

Morphology of phages by transmission electron microscope

A total of 16 representative phages (8, 4 and 4 for BHRP, MHRP and NHRP) were selected for transmission electron microscopy (TEM) analysis based on their lytic profiles and sources (Fig. 5). The lytic profiles and detailed dimensions of the phages are presented in Fig. 6.

Fig. 5
figure 5

Transmission electron microscopy images of isolated Listeria monocytogenes phages. Phages 17, 33, 263, 19, 225, 102, 62, and 251 belong to the Siphoviridae-like phage. Phages 211, 144, 201, 189, 130, 39, 208, and 222 belong to the Myoviridae-like phage family

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Fig. 6
figure 6

Lytic profiles and morphological characteristics of isolated Listeria monocytogenes phages. The numerical values within each cell represent the lysis ratio of each phage against the corresponding serotype strains. FE, Food-related environment; NE, Natural environment

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In the BHRP group, three phages were identified as Siphoviridae-like phages, with head diameters of 49.93 ± 0.94 nm and contractile tails measuring 6.48 ± 0.27 nm in diameter and 238.65 ± 17.50 nm in length. Furthermore, five phages were classified as Myoviridae-like phages, exhibiting head diameters of 78.52 ± 4.99 nm, contractile tails with diameters of 22.80 ± 1.76 nm, and lengths of 168.78 ± 19.92 nm.

In the MHRP group, two phages were identified as Myoviridae-like phage, with a head diameter of 77.32 ± 1.43 nm, a contractile tail diameter of 21.46 ± 2.04 nm, and a length of 187.13 ± 4.57 nm. Two phages were classified as Siphoviridae-like phages, had head diameters of 56.97 ± 0.26 nm, contractile tail diameters of 9.74 ± 1.29 nm, and tail lengths of 253.56 ± 1.69 nm.

In the NHRP group, four phages were identified as Siphoviridae-like phages, with head diameters of 54.39 ± 2.92 nm, contractile tails of 7.97 ± 0.84 nm in diameter, and lengths of 246.22 ± 12.62 nm (Fig. 6).

Discussion

Listeriosis is a severe foodborne illness with high fatality rates. It can result in miscarriage, spontaneous preterm labor, preterm birth, stillbirth, and congenital neonatal infections [1, 22]. Phages, as natural antibacterial agents, demonstrate considerable potential for the control of foodborne pathogens and the treatment of infectious diseases [16, 23, 24]. It is important to investigate the interactions between phages and Listeria to uncover the molecular mechanisms that could lead to the development of innovative antibacterial strategies. This study first conducted the large-scale endeavor to isolate Listeria phages from a multitude of sample environmental sources and diverse provinces, with 317 phages isolated. The host range of these phages was determined using a liquid culture method, which permitted the exploration of their lytic patterns and ecological distribution based on intraspecies serotype classification. Furthermore, their morphological characteristics were observed.

Several studies have provided insights into the morphology and distribution and abundance of phages in marine and soil environments. However, there is a lack of research isolating phages and characterizing their lytic activities [25]. Listeria-specific phages have been isolated from a variety of sources, including feces, wastewater, abattoir effluents, soil, farms, food products, and sewage [17, 18, 26,27,28,29,30,31]. Of the 500 identified Listeria phages, only a few have been fully characterized as virulent phages with potential for use in biological control [17, 19, 32,33,34]. These virulent Listeria phages have the capacity to infect a range of major L. monocytogenes serotypes (1/2a, 1/2b, 1/2c, 4a, 4ab, 4b, 4c, 4d, 4e) and Listeria innocua serotypes 5, 6a, and 6b. To date, no Listeria phages have been identified that are capable of lysing L. monocytogenes serotypes 3a, 3b, 3c, or Listeria grayii [16, 31]. Notably, several phages in this study were observed to lyse L. monocytogenes serotypes 3a and 3b strains.

Currently, there is no established reference standard for the lytic activity of phages. Furthermore, the categories such as “broad spectrum” and “narrow spectrum” lack clearly defined cutoff values. Using such terms is inherently subjective and provides limited comparability due to the relatively small sample sizes involved. This study inaugural attempt to categorize phages into three groups: broad host range phages (BHRP), medium host range phages (MHRP), and narrow host range phages (NHRP) based on total lysis ratios. This classification provides a comprehensive characterization of each group and elucidates the ecological roles of phages.

The vast majority of host strains, including those frequently associated with food contamination and human listeriosis, demonstrated susceptibility to BHRP phages in this study, this highlights the potential for their use in biocontrol and phage therapy applications. Further investigation is required to elucidate the resistance mechanisms observed in a few strains. Although L. monocytogenes is a well-known foodborne pathogen, 25 of the 33 BHRP phages in this study were isolated from the natural environment. This finding underscores natural environments as a significant reservoir of broad-spectrum phages.

The data indicate that phages in the NHRP group are capable of lysing serotype 4 strains, suggesting that serotype 4 strains exhibit greater ease of identification and lysis by phages. Therefore, serotype 4 strains are the most suitable for isolating Listeria phages. An additional potential explanation is the presence of variations in cell wall teichoic acids (WTA) between different serotypes of L. monocytogenes. Serotype 4 has WTA with terminal glucose and galactose residues, which are essential for phage adsorption and host lysis [35]. It is noteworthy that the majority of mitomycin C-induced Listeria phages (30 out of 39) were capable of lysing hosts of L. monocytogenes serotype 4 [36].

The lysis rates of BHRP phages were observed to be consistently high, while those of NHRP phages were found to be low across all cases in this study. In the MHRP group, food-related environment-sourced phages demonstrated a higher lysis rate against 1/2b strains than natural environment-sourced phages. Furthermore, the results also revealed that 1/2b strains exhibited greater susceptibility to phages derived from food-related environments than those from natural environments. These findings suggest that food-related environments may better support the survival and proliferation of 1/2b strains, aligning with previous studies reporting a high abundance of serotype 1/2b among Listeria monocytogenes isolates from food and food-related environments [8, 37,38,39,40,41]. To the best of our knowledge, there is no existing comparative study on isolates from both food-related environmental and natural environmental sources. Current research on L. monocytogenes primarily focus on isolates from food-related environmental sources and patients. In contrast, studies on natural environmental isolates are limited and often lack consistency [42,43,44]. The findings in this study offer insights into the distribution and ecological adaptability of Listeria in diverse environments.

It should be noted that this study has few limitations. The experimental design was not sufficiently comprehensive to permit exhaustive measurement of the host range. Accordingly, a diverse range of strains encompassing nine most prevalent serotypes was selected to ensure the attainment of representative results. It is conceivable that the host range may undergo alterations over time due to the co-evolution of phages and bacteria during successive propagation. This study describes the host range and morphology of the isolated Listeria phages. Further research is needed to evaluate the potential for more sophisticated applications, elucidate the mechanisms of bacterial resistance to phage, and gain a deeper understanding of the interactions between phages and hosts.

Conclusion

A total of 317 Listeria phages were isolated from a diverse array of sources in this study. The lysis patterns for nine serotypes of host strains and the ecological distribution of these phages were analyzed. The phages were classified into three groups based on their total lysis ratios. The majority of phages in the BHRP group are of the Myoviridae-like phages and are capable of lysing the majority of host strains, with minimal resistance observed. The phages of the MHRP group include both Siphoviridae-like and Myoviridae-like phages. Furthermore, phages isolated from food-related sources demonstrated greater lytic activity against Listeria serotypes 1/2b, 4a, and 4c compared to those derived from natural environmental sources. The phages in the NHRP group are of the Siphoviridae-like phages and are primarily capable of lysing serotype 4 strains. This study offers a valuable resource for the application of Listeria phages and provides new insights into the ecological distribution patterns of Listeria phages based on their lytic profiles.

Data availability

Not applicable.

Code availability

Not applicable.

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Acknowledgements

The authors thank Fenxia Fan for her excellent technical consultation.

Funding

This study was supported by grants from the National Institute for Communicable Disease Control and Prevention, China CDC (2021ZZKT003).

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Authors and Affiliations

  1. National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China

    Jinni chen, Yan Wang, Lingyun Liu, Hao Zhou, Pan Mao, Lingling Li, Ji Pu, Xuefang Xu, Jing Yang, Hui Sun, Xia Luo & Changyun Ye

  2. National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China

    Jingdong Song

  3. Research Center for Reverse Etiology, Workstation of Academician, Shanxi Medical University, Taiyuan, China

    Kui Dong

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  1. Jinni chen
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Contributions

JNC, CYY and YW conceived of the study. LLL (Lingyun Liu), PM, LLL (Lingling Li), XFX, JY, XL, HS, KD, and JDS, performed the experiment; HZ and JP analysed the data; JNC drafted the manuscript. CYY revised the manuscript. All authors reviewed and approved the final manuscript.

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Correspondence to Changyun Ye.

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chen, J., Wang, Y., Liu, L. et al. Isolation and relationship analysis of Listeria phages with various serotype hosts and morphological characterization. Virol J 22, 104 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12985-025-02706-w

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Virology Journal

ISSN: 1743-422X