The Viable But Non-Culturable State of Listeria monocytogenes in the One-Health Continuum

Abstract

Many bacterial species, including several pathogens, can enter a so-called "viable but non-culturable" (VBNC) state when subjected to stress. Bacteria in the VBNC state are metabolically active but have lost their ability to grow on standard culture media, which compromises their detection by conventional techniques based on bacterial division. Under certain conditions, VBNC bacteria can regain their growth capacity and, for pathogens, their virulence potential, through a process called resuscitation. Here, we review the current state of knowledge of the VBNC state of Listeria monocytogenes (Lm), a Gram-positive pathogenic bacterium responsible for listeriosis, one of the most dangerous foodborne zoonosis. After a brief summary of characteristics of VBNC bacteria, we highlight work on VBNC Lm in the environment and in agricultural and food industry settings, with particular emphasis on the impact of antimicrobial treatments. We subsequently discuss recent data suggesting that Lm can enter the VBNC state in the host, raising the possibility that VBNC forms contribute to the asymptomatic carriage of this pathogen in wildlife, livestock and even humans. We also consider the resuscitation and virulence potential of VBNC Lm and the danger posed by these bacteria to at-risk individuals, particularly pregnant women. Overall, we put forth the hypothesis that VBNC forms contribute to adaptation, persistence, and transmission of Lm between different ecological niches in the One-Health continuum, and suggest that screening for healthy carriers, using alternative techniques to culture-based enrichment methods, should better prevent listeriosis risks.

Keywords: VBNC; asymptomatic infections; dormancy; foodborne pathogen; infectious diseases; pregnancy; risk assessment; zoonosis.

Figure 1

Evidence for an intracellular VBNC state of L. monocytogenes (adapted from (Kortebi et al., 2017). (A) Simplified diagram of the different phases of the intracellular life of Lm in epithelial cells. Bacteria are internalized into the host cell and are contained in an entry vacuole. After escape into the cytosol, bacteria multiply and produce ActA, which allows them to polymerize actin (F-actin), move into the infected cell, and spread to adjacent cells (not shown). After a few days of infection, cytosolic bacteria cease to produce ActA and are captured in membrane compartments, forming Listeria-containing vacuoles (LisCVs) marked with LAMP1 (represented by red stars). In these acidic vacuoles, a subpopulation of bacteria can resist degradation and multiply slowly, up to entry into dormancy. (B, C) Observation of LisCVs by epifluorescence microscopy in human placental JEG3 cells infected for 3 days with Lm. LisCVs labelled with LAMP1 are in red, Listeria in green, DNA (stained with DAPI) in blue, and F-actin in white. (B) A cell contains several perinuclear LisCVs. The arrow indicates a LisCV shown at higher magnification in the image on the right. Bars: 2 μm. (C) LisCVs are present in mitotic cells infected with either the wild-type Lm strain (WT, left) or a ΔactA mutant (right). Bars: 2 μm. White arrows indicate representative LisCVs. (D–F) Subculturing of JEG3 cells infected with ΔactA bacteria leads to VBNC bacteria. (D) Cells infected for 3 days were subcultured and propagated until day 13 (d13) and stained as in (C). Intracellular bacteria in LAMP1-positive compartments are indicated by arrows. At the same time, plating of infected cell lysates onto agar plates produces no colony (not shown). (E) High magnification images of non-culturable LAMP1-positive bacteria at d13. A bacterium with a division septum (s) is shown on the right. Bar: 5 μm. (F) JEG3 cells infected with non-culturable bacteria were permeabilized with 0.1% Triton X-100 and stained with SYTO9 and PI (see BacLight assay, Table 1 ). Intact VBNC bacteria are stained green (arrow), while damaged bacteria (*) and nuclei are stained red. “Ph.C.”: phase contrast. Bar: 1 μm. The top right squared images show higher magnifications, with one bacterium with an intact membrane (green) and three bacteria with a compromised membrane (red).

Figure 2

The VBNC state of L. monocytogenes in the One Health continuum. (1) Lm is present in a wide range of environmental ecosystems (e.g., sea, rivers, lakes, soil, plants, fodder, and possibly within unicellular eukaryotes), either in a vegetative culturable state (VC) or in a viable but non-culturable (VBNC) dormant state that allows long-term survival under hostile conditions. (2) VC and VBNC bacteria are ingested by animals, in particular farmed and wild mammals, via food or water. (3) VBNC forms could be reactivated into VC forms in the gut by a resuscitation process. Active VC forms can lead to invasive infection or enter a steady state with the host, resulting in long-term asymptomatic carriage. Entry of bacteria into the VBNC state could contribute to this asymptomatic carriage, by promoting silent colonization of the gastrointestinal tract (gut-liver axis) or the female reproductive system. Resuscitation of dormant VBNC Lm in an asymptomatic host would reactivate a latent infection, leading to listeriosis or, if the infection is controlled by the host, to shedding of Lm into the environment. (4) Excretion of Lm from animals, in feces, abortive products or cadavers, releases the pathogen into the environment, initiating a new cycle. (5) During processing of animal or plant products, Lm can contaminate food industry production lines. Antimicrobial treatments and/or biofilm formation favor the appearance of VBNC bacteria and their persistence on surfaces, process wash waters, or in food matrices. VBNC Lm can thus contaminate raw or ready-to-eat foods. (6) Humans become infected by eating foods contaminated with VC or VBNC Lm. (7) As in animals, resuscitation of VBNC Lm in the gut might cause invasive listeriosis, mainly in at-risk populations (pregnant women, the elderly or immunocompromised persons). The VC Lm forms might also switch to the VBNC state in tissues, resulting in asymptomatic colonization of healthy carriers or a latent infection phase in at-risk individuals. (8) Human shedding of Lm releases the pathogen into the environment.