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Can Listeria monocytogenes endotoxin act as an A-B toxin?

Can Listeria monocytogenes endotoxin act as an A-B toxin?


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I think no, but I am not sure since Listeria is Gram-positive and probably has lipopolysaccharide (exception among Gram positive bacteria).

Can Listeria monocytogenes' endotoxin act like exotoxin A-B? (In which component B lets component A inside the cell and component A causes the toxicity.)


Considering great research we did after the answer was accepted, I should add important information about the topic. The old (and accepted) answer follows in "" marks for the sake of clarity.

We found that in spite of the earlier reports, the newest ones failed to find endotoxin-like structure in the Listeria born material. Most academic discussions about the issue say there is no such thing called "listeric endotoxin", thus I gave a right answer to a "compromised" question.

I should say that question is still brilliant because it promoted great research and should be an important source for everyone looking for information about listeric "endotoxin"

Listeria endotoxin is not a protein. Thus, is lacks enzymatic activity and acts indirectly by binding to sensitive receptors on macrophages. Thus, it can not cause lysis itself.

Considering AB-exotoxins, A-subunit is a protein that has enzymatic activity (responsible for toxin action) and subunit B has no enzymatic activity (protein responsible for binding to receptors).

Thus, the answer to your brilliant question is NO, Listeria endotoxin cannot act as AB-exotoxin being nonprotein and non enzymatic substance.

Also, don't be confused with Listeriolysin O, which is listeria's eXotoxin protein which is pore-creating protein.


Just to add some important relevant information to the answer from @Ilan.

There is no evidence for the presence of endotoxin/lipopolysaccharide in Listeria.

There was an early report of the presence of LPS (Wexler & Oppenheim, 1979) but this was contradicted by a later study (Maitra et al., 1986). The endotoxin activity of Gram negative bacteria is strongly associated with the lipid A moiety of the LPS. Lipid A is synthesised from N-acetylglucosamine in a three step pathway encoded by the genes lpxA, lpxC and lpxD. I obtained the sequences for the corresponding E. coli gene products and conducted BLAST searches against the Listeria monocytogenes genome with these results:

lpxA - best hit E=0.11

lpxC - no significant hit

lpxC - best hit E=1e-04

the last hit is to a 2,3,4,5-tetrahydropyridine-2,6-carboxylate N-succinyltransferase whereas the E. coli gene encodes a UDP-3-O-(3-hydroxymyristoyl)glucosamine N-acyltransferase. The similarity in the sequences seems to be because they are both members of a large superfamily of acyltransferases.

I conclude that Listeria monocytogenes does not not synthesise lipid A and therefore has no LPS, and no endotoxin.

Wexler, H., and J. D. Oppenheim. 1979. Isolation, characterization, and biological properties of an endotoxin-like material from the gram-positive organism Listeria monocytogenes. Infect.Immun. 23:845-857.

Maitra, SK et al. (1986) Establishment of beta-hydroxy fatty acids as chemical marker molecules for bacterial endotoxin by gas chromatography-mass spectrometry. Appl. Env. Mic. 52: 510-514


12.1.3: Virulence Factors

  • Contributed by OpenStax
  • General Biology at OpenStax CNX
  • Explain how virulence factors contribute to signs and symptoms of infectious disease
  • Differentiate between endotoxins and exotoxins
  • Describe and differentiate between various types of exotoxins
  • Describe the mechanisms viruses use for adhesion and antigenic variation

In the previous section, we explained that some pathogens are more virulent than others. This is due to the unique virulence factors produced by individual pathogens, which determine the extent and severity of disease they may cause. A pathogen&rsquos virulence factors are encoded by genes that can be identified using molecular Koch&rsquos postulates. When genes encoding virulence factors are inactivated, virulence in the pathogen is diminished. In this section, we examine various types and specific examples of virulence factors and how they contribute to each step of pathogenesis.


Overview of L. monocytogenes

          Scientists have been aware of Listeria monocytogenes since the 1920’s (4), yet much about it is still unknown. However, its life cycle has been studied carefully, as it is different from most pathogens. These differences lead to variations among effective treatment methods when infected with L. monocytogenes and other common gram-positive pathogens its specific pathogenesis is unlike most known. L. monocytogenes continues to be a hot topic for research because of its infamous lethality through infection (3). To effectively analyze the mechanisms used by hosts to ward off infection, one must first understand the life cycle and virulence factors of L. monocytogenes.


Abstract

Listeria monocytogenes is a food-borne pathogen responsible for a disease called listeriosis, which is potentially lethal in immunocompromised individuals. This bacterium, first used as a model to study cell-mediated immunity, has emerged over the past 20 years as a paradigm in infection biology, cell biology and fundamental microbiology. In this Review, we highlight recent advances in the understanding of human listeriosis and L. monocytogenes biology. We describe unsuspected modes of hijacking host cell biology, ranging from changes in organelle morphology to direct effects on host transcription via a new class of bacterial effectors called nucleomodulins. We then discuss advances in understanding infection in vivo, including the discovery of tissue-specific virulence factors and the 'arms race' among bacteria competing for a niche in the microbiota. Finally, we describe the complexity of bacterial regulation and physiology, incorporating new insights into the mechanisms of action of a series of riboregulators that are critical for efficient metabolic regulation, antibiotic resistance and interspecies competition.


Discussion

The pore-forming protein LLO is a primary determinant of L. monocytogenes pathogenesis that has a pronounced acid pH optimum and acts in an acidic vacuolar compartment to mediate escape of the bacterium into the host cytosol. In this study, we have examined the role of the acidic pH optimum in the pathogenesis of L. monocytogenes infection. By swapping residues with the less pH-sensitive ortholog PFO, we identified an LLO mutant, L461T, that was 10-fold more active at a neutral pH than wild-type LLO. L. monocytogenes synthesizing LLO L461T were 100-fold less virulent in mice than bacteria secreting the wild-type LLO, indicating that the acid pH optimum of LLO plays an important role during infection. Tissue culture assays revealed that bacteria synthesizing LLO L461T were not defective in the extent or pH of phagosomal escape, cell-to-cell spread, or in escape from the double-membraned vesicle formed during cell-to-cell spread. However, bacteria synthesizing LLO L461T permeabilized host plasma membranes, as shown by (a) gentamicin sensitivity of intracellular bacteria, (b) release of LDH from host cells, and (c) staining of DNA within infected cells with a membrane-impermeant dye. Thus, the results of this study strongly suggest that the acidic pH optimum of LLO is necessary to avoid damage to infected host cells.

LLO is unique among the CDCs in that its activity is normally repressed at a neutral pH. The biochemical basis of the LLO pH optimum is not understood it could reflect pH sensitivity in membrane binding, oligomerization, and/or pore formation. It was surprising that a single amino acid change, from leucine to threonine, could profoundly increase the hemolytic activity of LLO at a neutral pH. Neither leucine nor threonine alone contains properties that would suggest sensitivity to pH. The alteration from a nonpolar leucine to a polar threonine residue offers the potential of an additional hydrogen bond and a reduction in hydrophobicity, but in the absence of a detailed structure for LLO, it is difficult to determine the influence of these changes. However, if we assume a general structural similarity to PFO, because LLO and PFO are 42% identical (Rossjohn et al., 1997), then the L461T mutation is located on an outer loop of the fourth domain. Domain 4 has been implicated in both membrane binding and oligomerization (Nakamura et al., 1995 Bayley, 1997). Therefore, it is possible that the mutation may alter interactions with the target lipid bilayer or with other monomers in a pH-sensitive fashion. Also, there is evidence that domain 4 must undergo significant movement relative to the other domains when the monomer enters the lipid bilayer (Gilbert et al., 1999). In particular, the transmembrane hairpins of domain 3 have been proposed to pack with domain 4 when inserted in the membrane. The mutation of leucine 461 to threonine might affect intramolecular interactions in a manner that would make the cytolysin less sensitive to pH. The precise effect of the L461T mutation awaits additional biochemical analysis.

There are numerous examples of pathogens exploiting acidified compartments via pH-dependent proteins, including the viral hemagglutinins (Hernandez et al., 1996) and bacterial and protozoal toxins (Ley et al., 1990 Falnes and Sandvig, 2000). These precedents led us to hypothesize that the acidic optimum of LLO provides a mechanism to coordinate optimal pore-forming activity with maturation of the phagosome to allow efficient phagosomal escape, and any perturbation of this coordination would result in less efficient escape. Consistent with this hypothesis, a number of studies have shown that bafilomycin A1, a pharmacological inhibitor of phagosomal acidification, blocks the escape of L. monocytogenes from a phagosome (Conte et al., 1996 Beauregard et al., 1997). Therefore, it was surprising to find that the bacteria synthesizing a mutant LLO more active at a neutral pH escaped from the phagosome with a similar efficiency and at the same pH as wild-type bacteria. These data suggest that though acidification of the phagosome is important for bacterial escape, the pH sensitivity is not manifested through LLO activity. Given that bafilomycin A1 blocks maturation of endosomes (van Weert et al., 1995), these data suggest that a pH-dependent process other than LLO activity is involved in vacuolar perforation and escape.

Although the precise mechanism of vacuolar escape is not understood, there is recent evidence that CDCs, including LLO, may act as mediators of protein delivery to the host cytosol, similarly to the gram negative type III secretion system (Sibelius et al., 1996 Wadsworth and Goldfine, 1999 Madden et al., 2001). If indeed LLO acts to mediate cytosolic delivery from the phagosome, it is possible that the acid dependence is required for activation of some other pH-sensitive bacterial effector. At least one other L. monocytogenes virulence factor, a broad-range phospholipase C, is released upon vacuolar acidification (Marquis and Hager, 2000), so it is possible that LLO simply needs to form a pore to allow the transfer of the pH-sensitive effector to the cytosol to mediate vesicular escape. Another possibility is that a host factor involved in the escape process requires acidification to be activated or localized to the phagosome.

The most striking phenotype exhibited by L. monocytogenes expressing LLO L461T was the increased damage inflicted on the host cells' membranes. The simplest explanation for this observation is that LLO L461T is active within the neutral pH of the cytosol and passes a threshold at which the number of pores formed by the toxin overwhelms the host cell's membrane repair mechanisms (Walev et al., 2001). Alternately, it is possible that the mutation may lead to host cell damage through an unknown mechanism. However, our data clearly indicate that bacteria synthesizing LLO L461T permeabilize J774 host cells as early as 5 h after infection, whereas permeabilization by the wild type requires 8 h. It has been estimated that one bacterium secretes one LLO molecule per minute (Villanueva et al., 1995). Based on this rate and the doubling times for each strain, we calculated that at the time of permeabilization, the host cell is confronted with the production of ∼35 LLO L461T molecules per minute, enough monomers to assemble approximately one pore. In contrast, the wild-type bacteria are producing 420 LLO per minute when they permeabilize the host cell after 8 h of growth. These calculations indicate that bacteria synthesizing LLO L461T permeabilized the host cell with about one twelfth the production rate of wild type. This difference indicates that the unique acidic activity optimum of wild-type LLO enables L. monocytogenes to produce at least 10 times the number of progeny per infected host cell before significant host damage occurs.

Many factors contribute to the host cell's ability to deal with the presence of LLO within the cytosol. In light of studies suggesting that host cytosolic proteases cleave LLO (Villanueva et al., 1995 Decatur and Portnoy, 2000), an alternate explanation for the greater toxicity of LLO L461T is that it is less susceptible to proteolytic degradation. We observed an approximately twofold higher concentration of LLO L461T in the cytosol of J774 host cells, yet found that equal quantities of LLO were produced in vitro. Perhaps the greater concentration of LLO L461T reflected the fact that the cytolysin entered membranes more readily in the neutral pH of the cytosol where it was protected from proteolytic degradation, as has been shown for other CDCs (Nakamura et al., 1995). Regardless of the precise mechanism leading to the greater concentration of intracellular LLO L461T, we found that intracellular concentrations of LLO did not correlate with membrane permeabilization. A merodiploid LLO strain producing twice as much LLO was no more cytotoxic than the wild type, suggesting that a twofold increase in LLO concentration alone does not affect membrane damage under these conditions. However, a merodiploid strain synthesizing both the LLO L461T and the wild-type LLO acted similarly to the LLO L461T strain in the plaquing assay, producing gentamicin-sensitive plaques (unpublished data), suggesting that the mutant's cytotoxic phenotype is dominant over the wild type. These data suggest that the increased host membrane damage was due to the 10-fold greater activity of LLO L461T at a neutral pH and not simply intracellular LLO concentration.

The results of this study showed that L. monocytogenes expressing LLO L461T was ∼100-fold less virulent than the wild type. We hypothesize that the increased damage to the plasma membrane observed in tissue culture correlates with the defect in virulence in vivo. Additionally, we found that the LLO L461T grew less well in immunologically naive mice as early as 12 h after infection (unpublished data). Thus, the adaptive immune response probably plays little role in mediating the defect of the mutant bacteria. Instead, the defect is more likely to reflect the action of the innate immune response or a more basic defect in the bacterial life cycle in vivo. We can envision a number of scenarios that may lead to the mutant's virulence defect. One possibility is that a damaged cell no longer provides the advantages of an intracellular lifestyle, whether it is caused by the loss of access to nutrients, reduced actin-based motility, a loss of protection from humoral defenses, or another unknown mechanism. However, because the mutant grows and spreads normally at low gentamicin concentrations, a more likely possibility is that the mutant elicits a greater inflammatory response at the foci of infections due to greater cytotoxicity, thereby recruiting a greater number of leukocytes to the focus of infection. The hemolytic activity of LLO causes the production of inflammatory cytokines, leading to activation and chemotaxis of neutrophils and monocytes, which are largely responsible for containing the bacteria (Unanue, 1997 Kayal et al., 1999 Sibelius et al., 1999).

The results of this study support the concept that L. monocytogenes has evolved to minimize harm to its host cell. To achieve maximal virulence, the bacteria must maintain an equilibrium between producing a molecule that is cytolytic enough to mediate escape from the vesicle, yet not overly toxic to infected host cells. We hypothesize that L. monocytogenes has acquired a pathoadaptive mutation (Sokurenko et al., 1999) converting the primordial residue 461 to a leucine. This mutation is found solely in pathogenic Listeria and converts LLO from a toxin appropriate for an extracellular pathogen to that of an intracellular pathogen. Thus, LLO is highly active where it needs to be, in the acidic phagosome, yet relatively inactive in the neutral pH of the cytosol. Together with previously published data that LLO contains a PEST-like sequence that also reduces its toxicity (Decatur and Portnoy, 2000), our data imply that L. monocytogenes has evolved multiple failsafe mechanisms to regulate the activity of LLO, a molecule that has the potential to destroy its intracellular niche. Indeed, it has been hypothesized that the host cell uses these same acidic properties of the endocytic pathway to compartmentalize the activity of its own potentially hazardous lysosomal enzymes (Mellman et al., 1986).


S-Adenosylmethionine Metabolism and Aging

5.3 Types of Radical SAM Enzymes

At least a dozen types of radical SAM enzymes are involved in central metabolism and DNA repair, in the synthesis of many essential cofactors and nucleotide analogs that act as antibiotic, neoplastic agent, enhance translational fidelity, and synthesis of F420, the cofactor for hydride transfer in energy metabolism [6,172] ( Table 3.1 ). Spore photoproduct lyase can repair UV-induced thymine dimers in DNA in the absence of light [6] . Of clinical interest are antibiotics against drug-resistant bacterial pathogens, for example, Clostridium difficile, and toxins that may affect functioning of mitochondria and chloroplasts [6,173–175] . An example of a complex formation is the molybdene-containing “FeMo–Co” [MoFe7S9C] cluster in nitrogenase that catalyzes the reduction of nitrogen in our atmosphere to NH3 [6] . Well-known cofactors include modified tetrapyrroles such as (bacterio)chlorophyll, heme, and cobalamins [22] . Anaerobic ribonucleotide reductase reduces CTP to dCTP, a rate-limiting step in DNA metabolism strictly dependent on SAM [176] . The sulfur-inserting enzyme biotin synthase (BS/BioB) uses two FeS clusters in a difficult final step in the synthesis of vitamin B1 (essential in the methionine cycle). The reduced SAM-dependent [4Fe–4S] + cluster donates one electron to SAM producing a 5′-deoxyadenosine radical, which then requires a second half cluster, [2Fe–2S], to insert a sulfur atom into the biotin precursor [6,177,178] .


Can Listeria monocytogenes endotoxin act as an A-B toxin? - Biology

Bacterial Toxigenesis

Toxigenesis, or the ability to produce toxins, is an underlying mechanism by which many bacterial pathogens produce disease. At a chemical level, there are two main types of bacterial toxins, lipopolysaccharides, which are associated with the cell wall of Gram-negative bacteria, and proteins, which are released from bacterial cells and may act at tissue sites removed from the site of bacterial growth. The cell-associated toxins are referred to as endotoxins and the extracellular diffusible toxins are referred to as exotoxins.

Endotoxins are cell-associated substances that are structural components of bacteria. Most endotoxins are located in the cell envelope. In the context of this article, endotoxin refers specifically to the lipopolysaccharide (LPS) or lipooligosaccharide (LOS) located in the outer membrane of Gram-negative bacteria. Although structural components of cells, soluble endotoxins may be released from growing bacteria or from cells that are lysed as a result of effective host defense mechanisms or by the activities of certain antibiotics. Endotoxins generally act in the vicinity of bacterial growth or presence.

Exotoxins are usually secreted by bacteria and act at a site removed from bacterial growth. However, in some cases, exotoxins are only released by lysis of the bacterial cell. Exotoxins are usually proteins, minimally polypeptides, that act enzymatically or through direct action with host cells and stimulate a variety of host responses. Most exotoxins act at tissue sites remote from the original point of bacterial invasion or growth. However, some bacterial exotoxins act at the site of pathogen colonization and may play a role in invasion.

BACTERIAL PROTEIN TOXINS

Exotoxins are usually secreted by living bacteria during exponential growth. The production of the toxin is generally specific to a particular bacterial species that produces the disease associated with the toxin (e.g. only Clostridium tetani produces tetanus toxin only Corynebacterium diphtheriae produces the diphtheria toxin). Usually, virulent strains of the bacterium produce the toxin while nonvirulent strains do not, and the toxin is the major determinant of virulence (e.g. tetanus and diphtheria). At one time, it was thought that exotoxin production was limited mainly to Gram-positive bacteria, but clearly both Gram-positive and Gram-negative bacteria produce soluble protein toxins.

Bacterial protein toxins are the most powerful human poisons known and retain high activity at very high dilutions. The lethality of the most potent bacterial exotoxins is compared to the lethality of strychnine, snake venom, and endotoxin in Table 1 below.

TABLE 1. LETHALITY OF BACTERIAL PROTEIN TOXINS

Toxin
Toxic Dose (mg)
Host
Lethal toxicity
compared with:



Strychnine Endotoxin (LPS) Snake Venom
Botulinum toxin 0.8x10 -8 Mouse 3x10 6 3x10 7 3x10 5
Tetanus toxin 4x10 -8 Mouse 1x10 6 1x10 7 1x10 5
Shiga toxin 2.3x10 -6 Rabbit 1x10 6 1x10 7 1x10 5
Diphtheria toxin 6x10 -5 Guinea pig 2x10 3 2x10 4 2x10 2

Usually the site of damage caused by an exotoxin indicates the location for activity of that toxin. Terms such as enterotoxin, neurotoxin, leukocidin or hemolysin are descriptive terms that indicate the target site of some well-defined protein toxins. A few bacterial toxins that obviously bring about the death of an animal are known simply as lethal toxins, and even though the tissues affected and the target site or substrate may be known, the precise mechanism by which death occurs is not clear (e.g. anthrax LF).

Some bacterial toxins are utilized as invasins because they act locally to promote bacterial invasion. Examples are extracellular enzymes that degrade tissue matrices or fibrin, allowing the bacteria to spread. This includes collagenase, hyaluronidase and streptokinase. Other toxins, also considered invasins, degrade membrane components, such as phospholipases and lecithinases. The pore-forming toxins that insert a pore into eucaryotic membranes are considered as invasins, as well, but they will be reviewed here.

Some protein toxins have very specific cytotoxic activity (i.e., they attack specific types of cells). For example, tetanus and botulinum toxins attack only neurons. But some toxins (as produced by staphylococci, streptococci, clostridia, etc.) have fairly broad cytotoxic activity and cause nonspecific death of various types of cells or damage to tissues, eventually resulting in necrosis. Toxins that are phospholipases act in this way. This is also true of pore-forming hemolysins and leukocidins.

Bacterial protein toxins are strongly antigenic. In vivo , specific antibody neutralizes the toxicity of these bacterial exotoxins (antitoxin). However, in vitro, specific antitoxin may not fully inhibit their activity. This suggests that the antigenic determinant of the toxin may be distinct from the active portion of the protein molecule. The degree of neutralization of the active site may depend on the distance from the antigenic site on the molecule. However, since the toxin is fully neutralized in vivo , this suggests that other host factors must play a role in toxin neutralization in nature.

Protein exotoxins are inherently unstable. In time they lose their toxic properties but retain their antigenic ones. This was first discovered by Ehrlich who coined the term "toxoid" for this product. Toxoids are detoxified toxins which retain their antigenicity and their immunizing capacity. The formation of toxoids can be accelerated by treating toxins with a variety of reagents including formalin, iodine, pepsin, ascorbic acid, ketones, etc. The mixture is maintained at 37 degrees at pH range 6 to 9 for several weeks. The resulting toxoids can be used for artificial immunization against diseases caused by pathogens where the primary determinant of bacterial virulence is toxin production. Toxoids are effective immunizing agents against diphtheria and tetanus that are part of the DPT (DTP) vaccine.

Toxins with Enzymatic Activity

As proteins, many bacterial toxins resemble enzymes in a number of ways. Like enzymes, they are denatured by heat, acid and proteolytic enzymes, they act catalytically, and they exhibit specificity of action. The substrate (in the host) may be a component of tissue cells, organs or body fluid.

A plus B Subunit Arrangement

Many protein toxins, notably those that act intracellularly (with regard to host cells), consist of two components: one component (subunit A) is responsible for the enzymatic activity of the toxin the other component (subunit B) is concerned with binding to a specific receptor on the host cell membrane and transferring the enzyme across the membrane. The enzymatic component is not active until it is released from the native (A+B) toxin. Isolated A subunits are enzymatically active but lack binding and cell entry capability. Isolated B subunits may bind to target cells (and even block the binding of the native toxin), but they are nontoxic.

There are a variety of ways that toxin subunits may be synthesized and arranged: A + B indicates that the toxin is synthesized and secreted as two separate protein subunits that interact at the target cell surface A-B or A-5B indicates that the A and B subunits are synthesized separately, but associated by noncovalent bonds during secretion and binding to their target 5B indicates that the binding domain of the protein is composed of 5 identical subunits. A/B denotes a toxin synthesized as a single polypeptide, divided into A and B domains that may be separated by proteolytic cleavage.

Attachment and Entry of Toxins

There are at least two mechanisms of toxin entry into target cells.

In one mechanism called direct entry, the B subunit of the native (A+B) toxin binds to a specific receptor on the target cell and induces the formation of a pore in the membrane through which the A subunit is transferred into the cell cytoplasm.

In an alternative mechanism, the native toxin binds to the target cell and the A+B structure is taken into the cell by the process of receptor-mediated endocytosis (RME). The toxin is internalized in the cell in a membrane-enclosed vesicle called an endosome. H + ions enter the endosome lowering the internal pH which causes the A+B subunits to separate. The B subunit affects the release of the A subunit from the endosome so that it will reach its target in the cell cytoplasm. The B subunit remains in the endosome and is recycled to the cell surface.

In both cases above, a large protein molecule must insert into and cross a membrane lipid bilayer, either the cell membrane or the endosome membrane. This activity is reflected in the ability of most A+B or A/B toxins, or their B components, to insert into artificial lipid bilayers, creating ion permeable pathways. If the B subunit contains a hydrophobic region (of amino acids) that insert into the membrane (as in the case of the diphtheria toxin), it may be referred to as the T (translocation) domain of the toxin.

A few bacterial toxins (e.g. diphtheria) are known to utilize both direct entry and RME to enter into host cells, which is not surprising since both mechanisms are variations on a theme. Bacterial toxins with similar enzymatic mechanisms may enter their target cells by different mechanisms. Thus, the diphtheria toxin and Pseudomonas exotoxin A, which have identical mechanisms of enzymatic activity, enter their host cells in slightly different ways. The adenylate cyclase toxin of Bordetella pertussis (pertussis AC) and anthrax EF produced by Bacillus anthracis, act similarly to catalyze the production of cAMP from host cell intracellular ATP reserves. However, the anthrax toxin enters cells by receptor mediated endocytosis, whereas the pertussis adenylate cyclase traverses the cell membrane directly.

The specific receptors for the B subunit of toxins on target cells or tissues are usually sialogangliosides (glycoproteins) called G-proteins on the cell membrane. For example, the cholera toxin utilizes the ganglioside GM1, and tetanus toxin utilizes ganglioside GT1 and/or GD1b as receptors on host cells.

The best known and studied bacterial toxin is the diphtheria toxin, produced by Corynebacterium diphtheriae. Diphtheria toxin is a bacterial exotoxin of the A/B prototype. It is produced as single polypeptide chain with a molecular weight of 60,000 daltons. The function of the protein is distinguishable into two parts: subunit A, with a m.w. of 21,000 daltons, contains the enzymatic activity for inhibition of elongation factor-2 involved in host protein synthesis subunit B, with a m.w. of 39,000 daltons, is responsible for binding to the membrane of a susceptible host cell. The B subunit possesses a region T (translocation) domain which inserts into the endosome membrane thus securing the release of the enzymatic component into the cytoplasm.

Figure 1. Diphtheria Toxin (Dtx). A (red) is the catalytic domain B (yellow) is the binding domain which displays the receptor for cell attachment T (blue) is the hydrophobic domain responsible for insertion into the endosome membrane to secure the release of A. The protein is illustrated in its "closed" configuration.

In vitro, the native toxin is produced in an inactive form which can be activated by the proteolytic enzyme trypsin in the presence of thiol (reducing agent). The enzymatic activity of Fragment A is masked in the intact toxin. Fragment B is required to enable Fragment A to reach the cytoplasm of susceptible cells. The C terminal end of Fragment B is hydrophilic and contains determinants that interact with specific membrane receptors on sensitive cell membranes and the N-terminal end of Fragment B (called the T domain) is strongly hydrophobic. The specific membrane receptor for the B fragment has been shown to be a transmembranous heparin-binding protein on the susceptible cell's surface.

The diphtheria toxin enters its target cells by either direct entry or receptor mediated endocytosis. The first step is the irreversible binding of the C-terminal hydrophilic portion of Fragment B (AA 432-535) to the receptor. During RME, the whole toxin is then taken up in an endocytic vesicle. In the vesicle, the pH drops to about 5 which allows unfolding of the A and B chains. This exposes hydrophobic regions of both the A and B chains that can insert into the vesicle membrane. The result is exposure of the A chain to the cytoplasmic side of the membrane. There, reduction and proteolytic cleavage releases the A chain in the cytoplasm. The A fragment is released as an extended chain but regains its active (enzymatic) globular conformation in the cytoplasm. The A chain catalyzes the ADP ribosylation of elongation factor-2 (EF-2) as shown in Figure 2.


Figure 2. Entry and activity of diphtheria toxin (Dtx) in susceptible cells. The B domain of the toxin binds to a cognate receptor on a susceptible cell. The toxin is taken up in an endosome by receptor mediated encocytosis. Acidification of the endocytic vesicle allows unfolding of the A and B chains exposing the hydrophobic T domain of the toxin. The T domain inserts into the endosome membrane translocating the A fragment into the cytoplasm where it regains its enzymatic configuration . The enzymatic A component utilizes NAD as a substrate. It catalyzes the attachment of the ADP-ribose portion of NAD to elongation factor (EF-2) which inactivates its function in protein synthesis.

Table 2 describes several bacterial toxins with known enzymatic activity and the biological effects of the toxins in humans.

TABLE 2. BIOLOGICAL EFFECTS OF SOME BACTERIAL EXOTOXINS WITH ENZYMATIC ACTIVITY

TOXIN (subunit arr)* ENZYMATIC ACTIVITY BIOLOGICAL EFFECTS
Cholera toxin (A-5B) ADP ribosylates eucaryotic adenylate cyclase Gs regulatory protein Activates adenylate cyclase increased level of intracellular cAMP promote secretion of fluid and electrolytes in intestinal epithelium leading to diarrhea
Diphtheria toxin (A/B) ADP ribosylates elongation factor 2
Inhibits protein synthesis in animal cells resulting in death of the cells
Pertussis toxin (A-5B) ADP ribosylates adenylate cyclase Gi regulatory protein
Blocks inhibition of adenylate cyclase increased levels of cAMP affect hormone activity and reduce phagocytic activity
E. coli heat-labile toxin LT (A-5B) ADP ribosylates adenylate cyclase Gs regulatory protein Similar or identical to cholera toxin
Shiga toxin (A/5B Glycosidase cleavage of ribosomal RNA (cleaves a single Adenine base from the 28S rRNA)
Inactivates the mammalian 60S ribosomal subunit and leads to inhibition of protein synthesis and death of the susceptible cells pathology is diarrhea, hemorrhagic colitis (HC) and/or hemolytic uremic syndrome (HUS)
Pseudomonas Exotoxin A (A/B) ADP ribosylates elongation factor-2 analogous to diphtheria toxin
Inhibits protein synthesis in susceptible cells, resulting in death of the cells
Botulinum toxin (A/B) Zn ++ dependent protease acts on synaptobrevin at motor neuron ganglioside
Inhibits presynaptic acetylycholine release from peripheral cholinergic neurons resulting in flaccid paralysis
Tetanus toxin (A/B) Zn ++ dependent protease acts on synaptobrevin in central nervous system
Inhibits neurotransmitter release from inhibitory neurons in the CNS resulting in spastic paralysis
Anthrax toxin LF (A2+B) Metallo protease that cleaves MAPKK (mitogen-activated protein kinase kinase) enzymes

Combined with the B subunit (PA), LF induces cytokine release and death of target cells or experimental animals
Bordetella pertussis AC toxin (A/B) and Bacillus anthracis EF (A1+B)
Calmodulin-regulated adenylate cyclases that catalyze the formation of cyclic AMP from ATP in susceptible cells, as well as the formation of ion-permeable pores in cell membranes
Increases cAMP in phagocytes leading to inhibition of phagocytosis by neutrophils and macrophages also causes hemolysis and leukolysis
Staphylococcus aureus Exfoliatin B Cleaves desmoglein 1, a cadherin found in desmosomes in the epidermis
(also a superantigen)

Separation of the stratum granulosum of the epidermis, between the living layers and the superficial dead layers.

* toxin subunit arrangements: A-B or A-5B indicates subunits synthesized separately and associated by noncovalent bonds A/B denotes subunit domains of a single protein that may be separated by proteolytic cleavage A+B indicates subunits synthesized and secreted as separate protein subunits that interact at the target cell surface 5B indicates that the binding domain is composed of 5 identical subunits.

Pore-forming toxins, as the name suggests, insert a transmembranous pore into a host cell membrane, thereby disrupting the selective influx and efflux of ions across the membrane. This group of toxins includes the RTX toxins of Gram-negative bacteria, streptolysin O produced by S. pyogenes, and S. aureus alpha toxin. Generally, these toxins are produced as subunits that self-assemble as a pore on the eucaryotic membrane.

S. aureus alpha-toxin is considered the model of oligomerizing pore-forming cytotoxins. The alpha-toxin is synthesized as a 319 amino acid precursor molecule that contains an N-terminal signal sequence of 26 amino acids. The secreted mature toxin, or protomer, is a hydrophilic molecule with a molecular weight of 33 kDa. Seven toxin protomers assemble to form a 232 kDa mushroom-shaped heptamer comprising three distinct domains. The cap and rim domains of the heptamer are situated at the surface of the plasma membrane, while the stem domain serves as a transmembranous ion channel through the membrane.

TABLE 3. SOME PORE-FORMING BACTERIAL TOXINS

Toxin
Bacterial source
Target
Disease
perfringiolysin O
Clostridium perfringens
cholesterol
gas gangrene
hemolysin
Escherichia coli
cell membrane
UTI
listeriolysin
Listeria monocytogenes
cholesterol
systemic meningitis
anthrax EF
Bacillus anthracis
cell membrane
anthrax (edema)
alpha toxin Staphylococcus aureus
cell membrane
abcesses
pneumolysin
Streptococcus pneumoniae
cholesterol
pneumonia otitis media
streptolysin O
Streptococcus pyogenes
cholesterol
strep throat
leukocidin
Staphylococcus aureus phagocyte membrane
pyogenic infections

Superantigens: Toxins that Stimulate the Immune System

Several bacterial toxins can act directly on the T cells and antigen-presenting cells of the immune system. Impairment of the immunologic functions of these cells by toxin can lead to human disease. One large family of toxins in this category are the so-called pyrogenic exotoxins produced by staphylococci and streptococci, whose biological activities include potent stimulation of the immune system, pyrogenicity, and enhancement of endotoxin shock.

Pyrogenic exotoxins are secreted toxins of 22 kDa to 30 kDa, and include staphylococcal enterotoxins serotypes A-E, G, and H group A streptococcal pyrogenic exotoxins A-C staphylococcal exfoliatin toxin and staphylococcal TSST-1.

In general, the potent immunostimulatory properties of superantigens are a direct result of toxin binding to distinct regions outside the peptide binding cleft of the major histocompatibility class II molecules (MHC II), expressed on the surface of antigen-presenting cells, and to specific Vß elements on the T-cell receptor of T-lymphocytes. This results in a massive proliferation of up to 20% of peripheral T cells. Concomitant to T-cell proliferation is a massive release of cytokines from lymphocytes (e.g. interleukin-2, tumor necrosis factor ß, gamma interferon) and monocytes (e.g. IL-1, IL-6, tumor necrosis factor a). These cytokines serve as mediators of the hypotension, high fever, and diffuse erythematous rash that are characteristic of toxic-shock syndrome.

The staphylococcal enterotoxins are superantigens, but it is not known if this activity contributes to vomiting or diarrhea characteristic of staphylococcal food poisoning.

Control of Synthesis and the Release of Protein Toxins

The regulation of synthesis and secretion of many bacterial toxins is tightly controlled by regulatory elements that are sensitive to environmental signals. For example, the production of diphtheria toxin is totally repressed by the availability of adequate amounts of iron in the medium for bacterial growth. Only under conditions of limiting amounts of iron in the growth medium does toxin production become derepressed. The expression of cholera toxin and related virulence factors (adhesins) is controlled by environmental osmolarity and temperature. In B. pertussis, induction of different virulence components is staggered, such that attachment factors are produced initially to establish the infection, and toxins are synthesized and released later to counter the host defenses and promote bacterial survival.

The processes by which protein toxins are assembled and secreted by bacterial cells are also variable. Many of the classic exotoxins are synthesized with an NH terminal leader (signal) sequence consisting of a few (1-3) charged amino acids and a stretch of (14-20) hydrophobic amino acids. The signal sequence may bind and insert into the cytoplasmic membrane during translation such that the polypeptide is secreted while being synthesized. The signal peptide is cleaved as the toxin (protein) is released into the periplasm. Alternatively, the toxin may be synthesized intracytoplasmically, then bound to a leader sequence for passage across the membrane. Frequently, chaperone proteins are required to guide this process. Some multicomponent toxins, such as the cholera toxin, have their subunits synthesized and secreted separately into the periplasm where they are assembled. In Gram-negative bacteria, the outer membrane poses an additional permeability barrier that a protein toxin usually has to mediate if it is to be released in a soluble form. It has been proposed that some Gram-negative exotoxins (e.g. E. coli ST enterotoxin) might be released in membrane vesicles composed of outer membrane components. Since these vesicles possibly possess outer membrane-associated attachment factors, they could act as "smart bombs" capable of specifically interacting with and possibly entering target cells to release their contents of toxin.

The genetic ability to produce a toxin, including regulatory genes, may be found on the bacxterial chromosome, plasmids and lysogenic bacteriophages. Sometimes they occur within pathogenicity islands. In any case, the processes of genetic exchange in bacteria, notably conjugation and transduction, can mobilize genetic elements between strains and species of bacteria. Horizontal gene transfer (HGT) of genes that encode virulence is known to occur between species of bacteria. This explains how E. coli and Vibrio cholerae produce a nearly identical diarrhea-inducing toxin, as well as how E. coli O157:H7 acquired ability to produce shiga toxin form Shigella dysenteriae . The intestinal tract is probably an ideal habitat for bacteria to undergo HGT with one another.

There is conclusive evidence for the pathogenic role of diphtheria, tetanus and botulinum toxins, various enterotoxins, staphylococcal toxic shock syndrome toxin, and streptococcal pyrogenic exotoxins. And there is good evidence for the pathological involvement of pertussis toxin, anthrax toxin, shiga toxin and the necrotizing toxins of clostridia, in bacterial disease. But why certain bacteria produce such potent toxins is mysterious and is analogous to asking why an organism should produce an antibiotic. The production of a toxin may play a role in adapting a bacterium to a particular niche, but it is not essential to the viability of the organism. Most toxigenic bacteria are free-living in nature and in associations with humans in a form which is phenotypically identical to the toxigenic strain but lacking the ability to produce the toxin.

A summary of bacterial protein toxins and their activities is given in Tables 4. Details of the mechanisms of action of these toxins and their involvement in the pathogenesis of disease is discussed in chapters with the specific bacterial pathogens.

For more information and references on bacterial toxins go to this website: Bacterial Toxins: Friends or Foes?


Use as a transfection vector

Because L. monocytogenes is an intracellular bacterium, some studies have used this bacterium as a vector to deliver genes in vitro. Current transfection efficiency remains poor. One example of the successful use of L. monocytogenes in in vitro transfer technologies is in the delivery of gene therapies for cystic fibrosis cases. [ 29 ]

Cancer vaccine

A live attenuated L. monocytogenes cancer vaccine, ADXS11-001, is under development as a possible treatment for cervical carcinoma. [ 30 ]


This toxin has been shown to be the key virulence factor in infection with C. perfringens the bacterium is unable to cause disease without this toxin. [1] Further, vaccination against the alpha toxin toxoid protects mice against C. perfringens gas gangrene. [2] As a result, knowledge about the function of this particular protein greatly aids understanding of myonecrosis.

The alpha toxin has remarkable similarity to toxins produced by other bacteria as well as natural enzymes. There is significant homology with phospholipase C enzymes from Bacillus cereus, C. bifermentans, and Listeria monocytogenes. [3] The C terminal domain shows similarity with non-bacterial enzymes such as pancreatic lipase, soybean lipoxygenase, and synaptotagmin I. [4]

The alpha toxin is a zinc metallophospholipase, requiring zinc for activation. First, the toxin binds to a binding site on the cell surface. The C-terminal C2-like PLAT domain binds calcium and allows the toxin to bind to the phospholipid head-groups on the cell surface. The C-terminal domain enters the phospholipid bilayer. The N-terminal domain has phospholipase activity. This property allows hydrolysis of phospholipids such as phosphatidyl choline, mimicking endogenous phospholipase C. The hydrolysis of phosphatidyl choline produces diacylglycerol, which activates a variety of second messenger pathways. The end-result includes activation of arachidonic acid pathway and production of thromboxane A2, production of IL-8, platelet-activating factor, and several intercellular adhesion molecules. These actions combine to cause edema due to increased vascular permeability. [3]


Significance and Characteristics of Listeria monocytogenes in Poultry Products

Listeria monocytogenes is one of the most common foodborne pathogens. Poultry meat and products are of the main vehicles of pathogenic strains of L. monocytogenes for human. Poultry products are part of the regular diet of people and, due to nutrient content, more content of protein, and less content of fat, gain more attention. In comparison with red meat, poultry meat is more economical. So, it had a greater rate of consumption especially in barbecue form in which the growth of bacterium is favored. Subtyping of L. monocytogenes isolates is essential for epidemiological investigation and for identification of the source of contamination. In the following review, the main facet of presence of L. monocytogenes in poultry will be discussed. Most pathogenic serotypes of L. monocytogenes were detected in different products of poultry meat. Unfortunately, these isolated pathogens had sometimes resistance to commonly used antibiotics which were used for treatment of human infection.

1. Characteristics of Listeria monocytogenes

Listeria spp. are small gram-positive rod (0.5–4 μm in diameter and 0.5–2 μm in length), non-spore-forming, facultative anaerobic, catalase-positive, and oxidase-negative organisms. Listeria has tumbling motility at 20–25°C due to peritrichous flagella. Based on somatic (O) and flagellar (H) antigens, 13 serotypes were identified in Listeria monocytogenes (L. monocytogenes) including 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7 [1]. With the aid of multiplex PCR assay, four major serovars of L. monocytogenes strains can be categorized into four distinct serogroups, IIa (serovars 1/2a, 1/2c, 3a, and 3c), IIb (1/2b, 3b, 4b,4d, and 4e), IIc (1/2c and 3c), and IVb (4b, 4d, and 4e) by targeting four marker genes [2]. Food or food production environment is commonly contaminated with serotypes 1/2a, 1/2b, 1/2c, and 4b. The optimum growth temperature of L. monocytogenes is 30–37°C, but it can survive between 0 and 45°C. L. monocytogenes can multiply at refrigerator temperatures, is resistant to disinfectants, and adheres to various surfaces [1]. Once introduced into the processing plants, it is able to survive and remain for a long period under adverse conditions [1]. In the food industry, L. monocytogenes is able to form biofilm which can act as a potential source of contamination [3]. L. monocytogenes is a widely distributed organism in nature, with main reservoirs of soil and forage. Moreover, it was isolated from healthy humans and animals or infected domestic and wild animals [4].

2. Listeriosis

L. monocytogenes is the main cause of foodborne listeriosis in humans. Rarely, foodborne infections were reported by L. ivanovii and L. seeligeri. Strains of L. monocytogenes have different pathogenic potential, as some strains are very virulent, whereas some of them are noninfectious agents [4, 5]. Determination of the pathogenic potential of L. monocytogenes is important from food safety and public health perspective [6]. Identification of virulent strains can be achieved through tracing some genes directly related to pathogenicity of L. monocytogenes [7]. L. monocytogenes enters into host cells by use of a family of surface proteins called internalins, especially InlA and inlB. Moreover, InlC and InlJ also participate in the postintestinal stages of L. monocytogenes infection [8]. Putative internalins of L. monocytogenes are encoded by inlC (lmo1786) and inlJ (lmo2821) genes. The etiologic organism of human’s listeriosis harbors inlJ (lmo2821) [9]. L. monocytogenes carries a pore-forming toxin named listeriolysin O (LLO) (a 58 KDa protein-encoded by hlyA gene) which is vital for virulence of the bacterium [4]. LLO lyses the membrane of the vacuole and finally assists the entrance of L. monocytogenes into the cytoplasm [4]. Several methods had been used to assess the virulence of L. monocytogenes. Some of them include mouse virulence assay, cell culture, and use of specific genes and proteins [8]. Table 1 shows some specific genes used to determine the virulence of L. monocytogenes isolates in poultry.

Foodborne listeriosis has three main clinical features, namely, meningitis, septicemia, and abortion. In healthy humans it can cause febrile gastroenteritis, but in susceptible persons (children, elderly, immune-compromised and pregnant women) it may lead to septicemia and meningitis [1].

Listeriosis is the fourth commonly zoonotic disease in Europe, with the annual incidence of 0.41 cases per 100,000 population [10]. In Asian countries, reports of listeriosis rarely exist due to the failure of detection or report. Also, it may be due to lower incidence rate or exclusion of listeriosis for differential diagnosis by clinicians. However, L. monocytogenes has been regarded as one of the etiological factors of spontaneous abortions and stillbirth in India [11].

People more than 65 years old and neonates had the highest rates of infection with L. monocytogenes [12]. Maternal transmission to newborns was reported in 79% of cases. Listeriosis has the highest case fatality rate among foodborne diseases [10]. Isolation of L. monocytogenes from different kinds of RTE foods made it a remarkable foodborne pathogen [13, 14].

3. Subtyping of L. monocytogenes

Due to diverse strains of L. monocytogenes, subtyping of isolates for population genetics, source tracking, and the epidemiologic investigation is crucial for control and prevention of listeriosis. Typing of L. monocytogenes is needed to identify the sources of contamination and investigate foodborne listeriosis outbreaks [15, 16]. Phenotypic and genotypic subtyping are the two main methods which were used by researchers. As a phenotypic method, serotyping is generally used for L. monocytogenes strains related to disease outbreaks. Due to the involvement of only three serotypes in listeriosis outbreaks and low discriminatory power of serotyping in distinguishing of serotypes 4a, 4b, and 4c, serotyping does not have enough power for subtyping of L. monocytogenes [16]. So, PCR-based subtyping procedure such as Random Amplification of Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR), Repetitive Extragenic Palindromes-PCR (REP-PCR), Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR), and Pulsed Field Gel Electrophoresis (PFGE) gain more attention these days. RAPD assay amplified some random region in the L. monocytogenes genomes which generate distinct patterns. RAPD is more cost effective and faster than other typing methods, especially for low number of strains. RAPD-PCR technique is one of the main methods for bacterial strain characterization [15–18]. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) is a highly reliable, simple, and economic method which is able to produce clear fingerprint in Listeria [19]. ERIC-PCR analysis can separate the isolates of the same serotype. Also, it is capable of differentiating L. monocytogenes isolates which were detected in one sample with similar serotype [18].

Restriction Fragment Length Polymorphisms (RFLP) amplified one or some of the housekeeping or virulence-associated genes (e.g., hly, actA, and inlA) of L. monocytogenes and then digested PCR products with restriction enzymes [16]. It needs a low copy number of DNA to perform the experiment [16]. But, it has a lower discriminatory power and should be used along with other subtyping techniques and it is also more expensive than RAPD assay [20]. One of the other methods of genotyping of L. monocytogenes isolates is Amplified Fragment Length Polymorphisms (AFLP) method. In AFLP, digestion of DNA of isolates was done with two restriction enzymes including EcoRI, MseI, or TaqI [16]. One of the main advantages of AFLP is the high discriminatory power of this test [20]. In contrast, the pitfall of this method is low precision in fragment sizes, which leads to lower reproducibility [16].

PFGE is a tool in which, by exposing large DNA fragment to changing electric field, isolates were subtyped. This technique was more discriminatory than AFLP, but is more time consuming, expensive, and labor intensive in comparison with AFLP [20].

L. monocytogenes also has some randomly dispersed, repetitive sequence elements, such as repetitive extragenic palindromes (REPs) of 35–40 bp with an inverted repeat. These regions provide some useful points for strain distinction of L. monocytogenes isolates. Using REP-PCR, the origin of isolates was identified. It has an equal level of discrimination to PFGE. So it is suggested as a suitable technique for rapid typing of these isolates [16].

4. Listeria in the Poultry

4.1. Prevalence of Listeria Spp. and L. monocytogenes in the Poultry

One of the main vehicles of Listeria is poultry flocks which can spread the organism into the environment and poultry carcass due to unhygienic practice [21]. Occasionally, Listeria was isolated from the feces of poultry and chicken. Listeria spp. were detected in various poultry products [13, 22–25]. According to other studies, 8% to 99% of poultry products were contaminated with Listeria spp. [13, 24, 26, 27].

L. monocytogenes has been previously reported from different poultry products from raw products to cooked ones [13, 22, 28–35]. Schäfer et al. (2018) reported the contamination rate of breast and thigh samples of chicken as 8.64 and 44.19%, respectively [36]. 12.7 % of turkey meat was positive for L. monocytogenes [37]. Table 2 shows the contamination rate of poultry meat and products with Listeria spp. and L. monocytogenes.

According to Table 2, raw poultry meat and products were more contaminated with L. monocytogenes than cooked ones.

4.2. Serotypes of Listeria monocytogenes in Poultry

Serotypes 1/2b and 3b (serogroup IIb) of L. monocytogenes were the predominant isolated serotypes (52.77%) in chicken carcasses in Iran, and IVa serogroup which contains 4a and 4c serotypes also was detected in 27.77% of chicken carcasses [6]. The most common serotype in poultry products in the USA [38] was the same. But in another study, serotype 4b has been reported as the most common serotype in poultry products which was detected in 44.9% of the samples, while the prevalence of serotype 1/2b was 10.2% [33].

The prevalence of serogroup IVb was 2.77% and 12.5% in chicken carcasses [6] and RTE foods, respectively [14]. Human listeriosis is mainly caused by 1/2a, 1/2b, and 4b serovars of L. monocytogenes. However, 4b serotype was not commonly found in foods [6].

Fresh packed turkey meat samples were contaminated with L. monocytogenes serotypes as follows: 4b (or 4d, 4e) (51.4%), 1/2a (or 3a) (27.0%), and 1/2b (or 3b) (21.6%) [39]. However, serotype 4b was frequently isolated from turkey meat and legs, while 1/2b was prevalent in turkey breast samples [39].

About 16.66% of the chicken carcasses sampled in Iran were contaminated with serogroup IIa containing 1/2a, 3a, 1/2c, and 3c serotypes [6]. Another serological study on poultry products reported 1/2a serotype in 40.8% and 1/2c serotype in 4.08% of samples [33]. In other studies, 1/2a serotype was the predominant serotype in poultry products of Portugal and Estonia [40, 41], while in Finland 1/2c was the major one [42].The identified serogroups in RTE foods were 1/2a, 3a and 1/2c, 3c with the rate of 65.6% and 21.9%, respectively [14]. Based on the above studies, poultry meat is a potential source of pathogenic serotypes of L. monocytogenes.

4.3. Antimicrobial Susceptibility of Listeria monocytogenes

Listeria spp. are resistant to antimicrobial agents due to widespread mobile genetic elements and conjugative transposons [33]. Twelve out of 36 L. monocytogenes isolates were sensitive to 11 tested antimicrobial agents [22]. None of the isolates had resistance to ampicillin and vancomycin [22]. Some researchers observed resistance to ampicillin in L. monocytogenes isolates, but all of their isolates were sensitive to vancomycin [33]. Zeinali et al. (2017) observed resistance to erythromycin in 52.77% of L. monocytogenes isolates but, in another study, it was reported in 15.2% of the isolates [33]. 8 out of 23 of L. monocytogenes isolates had resistance to erythromycin [37]. Resistance to penicillin is a common finding in a number of studies [22, 33, 37, 43]. Moreover, high susceptibility of L. monocytogenes to ampicillin and penicillin is also reported [22, 27, 44–46]. Tetracycline is an antimicrobial agent with frequent use in poultry farms and also the treatment of human’s infection. Resistance to this agent is always observed in L. monocytogenes [13, 22, 33, 47, 48]. A low number of isolates were resistant to gentamycin [22, 49]. Standard therapy of listeriosis is done by use of ampicillin or penicillin G together with an aminoglycoside such as gentamicin. The second line of treatment belongs to trimethoprim. Resistance to trimethoprim in L. monocytogenes contributes to the pIP823 plasmid. There is a high susceptibility to this agent among L. monocytogenes isolates from foods [22, 49]. Most of the L. monocytogenes isolates had multidrug resistance. Fortunately, they are mostly sensitive to commonly used antibiotics which were used to cure human listeriosis.

4.4. Typing of L. monocytogenes Isolates in Poultry

Isolates of the L. monocytogenes with the same RAPD cluster belonged to different serogroup [15, 54–56]. Four different clusters were distinguished among 26 isolates of L. monocytogenes from chicken carcasses through RAPD analysis with three different primers, namely, OPM-01, HLWL 74, and D8635 [57]. These 26 isolates of L. monocytogenes had 16 antibiogram patterns [57].

L. monocytogenes isolates with similar pulse-types were classified in the same cluster in the RAPD assay. They were also clonally related [14]. Different laboratories used RAPD test for subtyping of L. monocytogenes isolates [14, 58], including isolates from different poultry processing plants [58, 59].

Several isolates of RTE foods were typed by RAPD, although they were indistinguishable by REP-PCR [14]. Twenty-eight isolates of L. monocytogenes from chicken meat had 27 RAPD types. They were resistant to three or more antimicrobial agents [60].

Fifteen isolates of L. monocytogenes from ducks had three antibiogram patterns, five RAPD clusters, and three singletons. So, RAPD had a higher power in distinguishing isolates [58].

Chicken and human isolates of L. monocytogenes were classified in five clusters in RAPD assay [54]. All human isolates were categorized in one cluster [54]. These isolates had different serogroup [54]. It was a common finding in other studies [15, 55, 56, 61]. It may be due to amplification of unspecific loci in RAPD test [15]. Most genetic similarities were seen among isolates which had common sampling area [54]. The same RAPD cluster was seen in some Lactobacillus strains from common source [62]. Discrimination power of RAPD test is higher than serotyping [54, 56]. Isolates in the same RAPD profile had different serotypes and were detected in different areas [15, 32, 54, 55, 63, 64].

29 isolates and 5 reference strains of L. monocytogenes were grouped into 4 clusters and 1 singleton by REP-PCR [65]. There was a high genetic diversity among isolates. According to Shi et al. (2015), isolates belonging to the same serotype and origin had the same cluster in REP-PCR. 15 isolates of L. monocytogenes from ducks and their environments were typed by RAPD and REP. They were categorized in 5 clusters and 3 singletons, and 2 clusters and 3 singletons, respectively. This finding proposed the suitability of these tools for discrimination of strains [58]. Soni et al. (2012) also observed that clinical isolates of L. monocytogenes had similar ERIC and REP fingerprints but are quite different from the water and milk isolates [47]. Oliveira et al. (2018) found 12 pulsotypes among 38 isolates of L. monocytogenes [17]. 40 isolates of L. monocytogenes produced 10 different fingerprint profiles in ERIC-PCR. Similar fingerprint were seen for isolates of the same sample, but there was two strains in one sample with different fingerprints [18]. L. monocytogenes had a high genetic diversity, and for good differentiation of isolates the use of at least two subtyping approaches is necessary.

5. Conclusion

In conclusion, from the food safety perspective, the presence of L. monocytogenes in the poultry meat and products is a multifaceted potential hazard. This is due to, firstly, some barbecued and fried foods based on chicken meat which may lead to the survival of L. monocytogenes in final products and, secondly, the presence of multidrug resistance isolates which transfer the antibiotic resistance to community. Also, some of the isolates were pathogenic serotypes that play a major role in human listeriosis outbreaks. Subtyping data revealed the heterogeneous nature of the L. monocytogenes isolates. RAPD, REP-PCR, and ERIC-PCR have a considerable discriminatory power and are cost effective and less tedious and time consuming.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

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Copyright © 2019 Abdollah Jamshidi and Tayebeh Zeinali. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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