The Transcriptional Response of Listeria monocytogenes during Adaptation to Growth on Lactate and Diacetate Includes Synergistic Changes That Increase Fermentative Acetoin Production

Abstract

The organic acids lactate and diacetate are commonly used in combination in ready-to-eat foods because they show synergistic ability to inhibit the growth of Listeria monocytogenes. Full-genome microarrays were used to investigate the synergistic transcriptomic responses of two L. monocytogenes strains, H7858 (serotype 4b) and F6854 (serotype 1/2a), to these two organic acids under conditions representing osmotic and cold stress encountered in foods. Strains were exposed to brain heart infusion (BHI) broth at 7°C with 4.65% water-phase (w.p.) NaCl at pH 6.1 with (i) 2% w.p. potassium lactate, (ii) 0.14% w.p. sodium diacetate, (iii) the combination of both at the same levels, or (iv) no organic acids as a control. RNA was extracted 8 h after exposure, during lag phase, to capture gene transcription changes during adaptation to the organic acid stress. Significant differential transcription of 1,041 genes in H7858 and 640 genes in F6854 was observed in at least one pair of the 4 different treatments. The effects of combined treatment with lactate and diacetate included (i) synergistic transcription differences for 474 and 209 genes in H7858 and F6854, respectively, (ii) differential transcription of genes encoding cation transporters and ABC transporters of metals, and (iii) altered metabolism, including induction of a nutrient-limiting stress response, reduction of menaquinone biosynthesis, and a shift from fermentative production of acetate and lactate to energetically less favorable, neutral acetoin. These data suggest that additional treatments that interfere with cellular energy generation processes could more efficiently inhibit the growth of L. monocytogenes.Listeria monocytogenes is a psychrotolerant food-borne pathogen that is of particular concern to the ready-to-eat-meat and -seafood industries because its ability to grow at temperatures as low as Ϫ0.4°C (70) reduces the ability of refrigeration to control the pathogen's growth in foods. Because L. monocytogenes generally contaminates foods at low levels (27) and has a high infectious dose (69), the ability of the organism to grow in foods is critical for its ability to cause disease (16). Foods do not support growth if they are stored frozen, have a pH of Յ4.4, have a water activity of Ͻ0.92, or incorporate some type of growth inhibiting measure (21), e.g., chemical preservatives (32). Ready-to-eat meat and seafood products have been identified as high-risk foods for listeriosis, as these foods support growth of L. monocytogenes (22), but it has also been predicted that reformulation of U.S. deli meats with effective growth inhibitors, e.g., the organic acids lactate and diacetate, would result in a 2-to 8-fold reduction of listeriosis due to consumption of these products (55). One particular advantage to using the combination of lactate and diacetate for L. monocytogenes control in meat and seafood products is that these inhibitors have been shown to have greater-than-additive, i.e., synergistic, growth-inhibiting effects (65), although the mechanisms...

Transcriptomic Analysis of the Adaptation of Listeria monocytogenes to Growth on Vacuum-Packed Cold Smoked Salmon

“…The production of acetate and propionate as by-products of ethanolamine and 1,2-propanediol degradation suggests that growth inhibitors that include these two organic acids may be able to more effectively inhibit L. monocytogenes growth than currently used growth inhibitors. This is supported by a recent transcriptomic study in BHIB-grown L. monocytogenes (17), which showed that exposure of L. monocytogenes to acetate and lactate led to decreased expression of lactate-and acetate-creating energy pathways, shifting ATP production to a less efficient pathway with acetoin, a noncharged molecule, as an end product.…”

“…A number of previous studies have analyzed the transcriptomes of foodborne pathogens under stress conditions commonly present on/in food matrices (including hyperosmotic stress, cold stress, hydrostatic pressure stress, antimicrobial stress, acid stress, and alkali stress), using laboratory media modified to simulate these conditions as a model (15)(16)(17)(18)(19)(20)(21)(22)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63). These laboratory media include BHI broth (18,20,21,59), BHI agar (19), Luria-Bertani broth (60,61), tryptic soy broth (62), M9-glucose (63), and Listeria minimal medium (22) as reference conditions.…”

“…This will facilitate the identification of new compounds that could specifically interfere with these metabolic pathways and thereby control the growth of L. monocytogenes (14). Extensive studies on the transcriptome of L. monocytogenes have been conducted to assess how it responds to the physical, chemical, or biological stresses that it may encounter on/in food matrices (15)(16)(17)(18)(19)(20)(21)(22). The majority of data from these experiments are based on exposure of L. monocytogenes to specific stresses in laboratory media, providing information about specific stress responses and transcriptional profiles in a controlled environment.…”

VirR-Mediated Resistance of Listeria monocytogenes against Food Antimicrobials and Cross-Protection Induced by Exposure to Organic Acid Salts

“…Nevertheless, this potential cross-protective effect has not been assessed for other relevant food antimicrobial compounds. Further, the evidence of VirR regulon induction under acid stress prompted us to hypothesize that a potential cross-protective effect would be VirR mediated (43)(44)(45). Our results indicated that exposure to 2% PL increased subsequent NIS and EPL resistance of both the parent strain and the ⌬virR mutant.…”

“…In contrast, there have been reports of a cross-protective phenomenon, where prior exposure to a stress may increase resistance against a subsequent stress (20, 42). Additionally, acid-induced transcriptome profiling in L. monocytogenes has suggested upregulation of stress-and virulence-related genes, including the VirR regulon, as part of acid stress adaptation (43)(44)(45). To test the hypothesis that prior exposure to organic acids induces crossprotection against the selected antimicrobials and is mediated by VirR, we exposed the L. monocytogenes parent strain and H7858 ⌬virR to BHI (no acid control), 2% PL, and 0.14% SD, followed by subsequent challenge with antimicrobial compounds.…”