Listeria monocytogenes Preservative Resistance

Introduction

Growth of Listeria on PALCAM selective and differential agar. Black circles around colonies indicate esculin hydrolysis, a characteristic of Listeria and related species. Photo credit: Generon.

By Iris Pardue
Listeria monocytogenes is a facultatively anaerobic Gram-positive food pathogen of the phylum Bacillota capable of surviving and growing at low temperatures. As a result, it is responsible for infections in deli meats, cheeses, and other refrigerated products. Outbreaks are relatively frequent, and serious cases have an especially high fatality rate of 15.26% over recorded cases in the US in the last decade. Unsurprisingly, much research has been conducted into control of Listeria as a result. Many different methods are used by food supply companies to inhibit bacterial growth, and chief among these methods are chemical preservatives. There are several different types of preservatives commonly used, and many have different mechanisms of action that will be discussed later. Having a diverse portfolio of antimicrobial preservatives available is especially important given the ability of pathogens to quickly evolve resistance to common mechanisms. This article explores the variety of commonly used preservatives relevant to controlling listeriosis along with the susceptibilities and resistances possessed by Listeria to each.

Cold Resistance

The anteiso-17-0 and anteiso-15-0 fatty acids, respectively at top and bottom. Photo credit: Pubchem and Pubchem

To fully understand the resistance of Listeria to preservatives, it is important to first consider its most importance resistance to cold. Listeria is a psychrophile, capable of growing at freezing temperatures as well as at human body temperatures. Numerous mechanisms exist to aid resistance to cold, but chief among them is adaptations in the content of the phospholipid membrane. This adaptation is mainly achieved via the use of the two primary fatty acids in the lipid membrane, anteiso-17-0 and anteiso-15-0. The proportion between these two fatty acids is modulated in response to low temperatures until the more flexible anteiso-15-0 dominates, reaching 80% of the total fatty acid profile. This adaptation allows it to maintain the crucial "liquid-crystal" state, which is necessary for cell function by lowering the melting point of the membrane. However, the focus on lipids alone can paint an oversimplified picture of the cell membrane's temperature stability. In many species, cholesterol acts as a cellular antifreeze to aid membrane stability, and Listeria employs a similar mechanism using isoprenoid quinones. Menaquinone-7 (MK-7), also known as vitamin K2, is composed of a napthaquinone ring fixed to a long chain of 7 isoprene units, which allows it to integrate into the membrane, a structure shared with the quinones used in electron transport. Flegler et al. found that strains containing higher levels of these molecules in their membranes better survived temperature stresses and did not adapt their fatty acid composition to the same degree as other strains. However, product feedback inhibition of the shikimate pathway responsible for synthesis of aromatic amino acids that are processed into quinones did reduce the ability of high MK-7 strains to adapt to the lower temperatures, providing a possible target for future preservatives. Indeed, a drug targeting chorismate synthase, an enzyme that catalyzes a crucial transformation in the shikimate pathway reportedly showed promising antimicrobial activity against Listeria and other species, and could be developed into a future food additive.

Acid Preservatives

The EVOH copolymer structure. Hydroxyl side chains from vinyl alcohol monomers are interspersed randomly along the chain based on the proportion of vinyl alcohol used in the polymerization. Photo credit: Wikimedia Commons.

A large number of food preservatives are organic acids capable of antimicrobial activity while maintaining levels safe for human consumption, such as sorbic acid, benzoic acid, lactic acid, and ascorbic acid. It is generally accepted that they pass easily through the membrane as neutral, protonated molecules that dissociate to toxic anions within the cell that exhibit a variety of antimicrobial effects; however, transport of organic acids via anion channels is also possible. Additionally, the change in external pH induced by these preservatives can force Listeria to activate its acid tolerance responses. Studies have found that proton stress has a notable effect on the C15:C17 ratio of the membrane, potentially compromising the methods used to resist colder environments. Use of organic acids in particular resulted in numerous additional membrane changes, including a further decrease in the C15:C17 ratio and a higher level of C18 fatty acids that could further compromise the cold stress response. However, these results were collected at pH 6.0, which is well above the pKa of most organic acids used for preservatives. As a result, these acids were primarily in their anionic state in which they cannot pass the membrane, and the "toxic anion" effect may not be as intense as it is in foods like yogurt that primarily exist at pH 4.6 or below. Further research at lower pH might reveal a more intense version of these same effects or perhaps a different set of effects entirely. A different study conducted at pH 5.5 revealed that the membrane changes induced by organic acid stress do in fact increase the melting point of the membrane, possibly affecting its ability to remain in a liquid-crystal state. However, from the resilience of Listeria to continue growing on acidic substrates in refrigerated environments, there is evidently some mechanism of resistance to acid that allows it to survive, despite potential membrane adaptations that could harm its cold resistance. Listeria does not form spores, and thus cannot stay dormant in these conditions in a similar fashion to other Bacillota. Indeed, research indicates that Listeria is capable of habituating to acids, a trait necessary for both survival in acidic foods and infecting the human gut. However, the mechanisms of action of the toxic organic anions are yet to be elucidated in detail.

Soluble or volatile acids are not the only way for these acids to exercise an antimicrobial effect. The active ethylene vinyl alcohol copolymer (EVOH) is already widely used in food preservation as a gas-impermeable layer that prevents oxidation of a food and inhibits aerobic microbe growth This alone is not enough to impede Listeria growth due to its facultatively anaerobic nature, but research has been done on combining EVOH with other materials to enhance antimicrobial properties. Sorbic acid-chitosan microcapsules mixed evenly into 38% ethylene EVOH showed increased antimicrobial ability against E. coli, Salmonella enteriditis and Listeria, although the effect on Listeria was less pronounced. The sorbic acid was capable of diffusing into multiple food simulants over the course of several days, potentially extending the shelf life of Listeria-susceptible foods by a significant amount. A variety of different chemicals could be packaged into capsules in this way, which may open up effective antimicrobial agent delivery to new molecules or improve effectiveness of common organic acid preservatives.

Inorganic Preservatives

A diagram of the formation of peroxynitrite via reactions with metalloproteins in vivo. The porphyrins described here may not be the same as those present in Listeria, but the general concept remains the same. Photo credit: Szabó et al.

Nitrites are a common inorganic preservative in meat processing, often added as sodium nitrite (NaNO₂) during the curing process to reduce oxidation, limit microbe growth (especially of Clostridium), and provide the unique flavor of cured meats. However, their levels must be tightly controlled due to their formation of carcinogenic nitrosamines within the body and thus must be used in limited concentrations. Nitrite does reduce growth of Listeria on spreadable sausages at marketable concentrations, an effect that increased significantly with lower pH until growth was completely ablated at pH 5.0. When the bacteria were allowed to adapt, the transcriptome was largely modified to favor stress-related processes as opposed to central cellular processes such as membrane expansion, translation and genome repair. Interestingly, many of these genes are similar to those upregulated when the cells are phagocytosed by macrophages, an environment where oxidative and acidic stressors are common in the lysosome. The majority of differentially expressed genes were poorly characterized, which further highlights the need for more research on the mechanism of action of Listeria habituation to preservatives. The number of listeriosis infections has increased in recent years, which may be due to a developing resistance to traditional preservation methods like nitrite curing. Nitrite's mechanism of action is mainly due to highly oxidative radical forms of the molecule that exist via reactions with superoxide (a radical form of dioxygen) to form peroxynitrate. Listeria is capable of detoxifying superoxide with an oxygen- and salt-inducible superoxide dismutase, which would inhibit peroxynitrite formation. This combined with the ability of Listeria to grow anaerobically where superoxide cannot activate nitrites to their radical forms suggests that it likely possess a strong innate resistance to nitrite, as evidenced by continued contamination of cured meats. This resistance could be provided by catalase or superoxide dismutase, both of which are known to be present in Listeria and contain metal ion centers that could catalyze a reaction to detoxify peroxynitrite, as suggested at right.

Stress Response Regulation

A schematic of sigma factor σ⁷⁰ binding to the core promoter of E. coli. Photo credit: Mark S. Paget.

In bacteria, the genetic response to stress is controlled by a variety of factors, but chief among these is the change in the sigma factor used to initiate transcription. In Listeria and other Bacillota, this is controlled mainly by the sigma b (σ^B) factor associated with its general stress response. σ^B, like all sigma factors, binds to the bacterial core promoter in order to recruit RNA polymerase by mediating the attachment between preserved DNA elements and the β' subunit of the polymerase, forming the RNA polymerase holoenzyme. A number of different sigma factors are expressed, but σ^B specializes in transcription of genes involved in the starvation and stress response. In Bacillus subtilis, it is involved in regulating the sporulation cascade, but presumably regulates a different group of genes in Listeria due to its lack of sporulation.. ΔsigB mutants are known to be more susceptible to cold and organic acid stresses, and a total of 198 genes show lower expression compared to wild-type strains grown at 3° C. The σ^B regulon appears to overlap largely with the σ^L regulon under these conditions, and a ΔsigBL double knockout mutant showed even higher susceptibility to the aforementioned stressors. Some biosynthetic genes such as components of the phosphoryltransferase system, purine ribonucleotide synthesis genes, and ribosomal genes remained downregulated at 3° C. It is unclear whether this is due to a loss of sigma factors or a result of other stress-related transcription factors slowing growth-related biosynthesis to preserve other functions. Interestingly, flagella-associated genes including flagellin were downregulated in ΔsigBL mutants, resulting in a significant loss of motility. Flagellar motility is known to be significantly upregulated in response to cold stress and aid survival, but why motility is necessary for cold resistance remains unknown. If these downstream stress response genes are to be targeted by drugs, their importance to survival must be much better characterized.

The idea of targeting sigma factors with antimicrobial drugs is not entirely new, and a screening method for cyclic peptides has been developed in E. coli. Remarkably, cyclic peptides have already been considered as potential food preservatives, opening the way for these screening methods to be used in development anti-Listeria food treatments. Sigma factors may be a particularly good target for antimicrobial drugs, as they cannot undergo significant changes of structure to reduce drug binding without risking loss of function due to the preserved nature of the core promoter. However, efflux methods or drug-degrading proteases could still emerge and reduce the effectiveness of cyclic peptide treatments.

Conclusion

In sum, there are a vast range of food preservation mechanisms that have varying effects on Listeria. Despite this, the rising rate of listeriosis outbreaks implies that our current status quo of preservation methods is not as effective as it needs to be. Further characterization of membrane fluidity regulation, organic acid mechanisms of action, peroxynitrite detoxification, flagellar role in cold resistance, and other stress response genes can elucidate new essential mechanisms for stress survival that can be targeted by new food additives. Without that research, the vulnerabilities of the stress response remain veiled to the eyes of researchers. Additionally, finding ways to overlap known stresses, such as in the acidified nitrite experiment, could reveal effective preservation methods that require minimal new development and research since they combine pre-validated preservatives. Highly resilient pathogens like Listeria pose a unique set of challenges, but with the right depth of research in the right areas, these challenges can be overcome and outbreaks can be more effectively controlled.

References

Authored for BIOL 238 Microbiology, taught by Joan Slonczewski,at Kenyon College,2024