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Article

Genetic Determinants Associated with Persistence of Listeria Species and Background Microflora from a Dairy Processing Environment

by
Vaishali Poswal
1,
Sanjeev Anand
1,*,
Jose L. Gonzalez-Hernandez
2 and
Brian Kraus
3
1
Department of Dairy and Food Science, South Dakota State University, Brookings, SD 57006, USA
2
Department of Agronomy, Horticulture and Plant Science, South Dakota State University, Brookings, SD 57006, USA
3
Wells Enterprises Inc., Le Mars, IA 51031, USA
*
Author to whom correspondence should be addressed.
Appl. Microbiol. 2026, 6(1), 20; https://doi.org/10.3390/applmicrobiol6010020
Submission received: 20 November 2025 / Revised: 15 January 2026 / Accepted: 16 January 2026 / Published: 21 January 2026
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Abstract

Listeria monocytogenes is a persistent foodborne pathogen capable of surviving in food processing environments, often in association with diverse environmental microflora. This study examines genomic determinants of persistence, specifically stress adaptation and biofilm-associated traits, in environmental Listeria species and other environmental microflora from a dairy processing facility by analyzing whole-genome sequences of 6 environmental Listeria isolates, 4 ATCC reference strains, and 22 air and floor swab cultures, annotated using the RAST platform. Subsystem analysis revealed that Listeria isolates carried a defined set of genes linked to biofilm formation, antimicrobial resistance, and stress response, though in lower abundance than environmental cultures. Listeria exhibited fewer flagellar genes but greater consistency in core stress-related genes, including those for disinfectant and osmotic stress resistance, with SigB operon and RpoN genes highlighting strong stress tolerance. In contrast, environmental cultures exhibited broader transcriptional regulators (RpoE, RpoH) and greater diversity in acid and heat shock response genes, indicating distinct survival strategies. All examined Listeria species harbor biofilm and stress-resistance genes enabling independent survival, while environmental microbiota show greater genetic diversity that may promote persistence and multispecies biofilm formation. This study underscores the complex genetic landscape that may contribute to the persistence of Listeria and environmental microbiota in dairy processing environments, providing foundational insights for environmental cross contamination control strategies.

1. Introduction

Listeria monocytogenes (Lm) is a Gram-positive, facultatively anaerobic foodborne pathogen of significant concern due to its ability to cause listeriosis, a severe invasive disease with high hospitalization and mortality rates, particularly among immunocompromised individuals, pregnant women, neonates, and the elderly [1,2]. Lm has been associated with a few foodborne outbreaks involving dairy products such as raw milk, soft cheese [3], and ice cream [4,5]. A recent outbreak in the United States, linked to contaminated supplement shakes, resulted in 41 hospitalizations and 14 deaths, underscoring the pathogen’s public health significance [6].
One of the primary factors contributing to the repeated emergence of Lm in foodborne outbreaks is its persistence in food processing environments (FPEs), where it can survive despite routine sanitation efforts [7]. The persistence of environmental Lm is largely attributed to its remarkable ability to withstand harsh physicochemical stressors commonly encountered in FPEs, such as cold temperatures, acidic pH, osmotic and oxidative stress, desiccation, nutrient limitation, and recurrent exposure to disinfectants [1,8]. In addition to these inherent tolerances, Lm persistence is enhanced by a variety of adaptive mechanisms, including the activation of stress response regulators and the capacity to form biofilms [9,10,11]. Biofilms enable Lm to adhere to surfaces and resist environmental stresses through the production of extracellular polymeric substances (EPS), which serve as a protective barrier against shear stress, desiccation, and sanitizers, thereby reducing the efficacy of sanitation procedures [8,11]. Genomic studies have identified several persistence-associated genetic determinants in Lm, including stress-related genes (sigB, clpC, gadD, grpE), biofilm-associated loci (flaA, prfA, actA, bapL), and quorum-sensing systems (agrBDCA, luxS) [11].
Although some previous research has elucidated the genomic mechanisms underlying Lm persistence [1,8,9,11,12], the influence of coexisting microbial consortia within FPEs remains insufficiently characterized. Environmental Lm frequently cohabitates with diverse background microbiota, which are increasingly recognized for their capacity to enhance persistence indirectly by facilitating multispecies biofilm formation [13]. These microbial communities modulate Lm phenotypes through strain-dependent interspecies interactions that affect survival, biofilm development, and stress tolerance. Such interactions, whether synergistic or antagonistic, may also promote horizontal gene transfer via mobile genetic elements, including plasmids, integrons, and transposons, thereby enabling the dissemination of persistence-associated traits across taxa [12,14]. The recurrent recovery of biofilm-forming, sanitation-resistant microorganisms from FPEs, including non-pathogenic Listeria spp. and diverse background microflora, following cleaning procedures, highlights the necessity for comparative genomic analyses encompassing both Lm and cohabiting microbiota to clarify their contributions to persistence and ecological adaptation [15]. However, most studies have disproportionately focused on virulent Lm strains, neglecting non-pathogenic Listeria species such as L. innocua (Li) and L. welshimeri (Lw), which are commonly co-isolated with Lm and may carry homologous or accessory genes involved in environmental persistence [16].
Whole-genome sequencing (WGS) serves as a powerful tool for characterizing genetic determinants that have been potentially associated with biofilm formation and persistence in environmental Listeria strains and the background microflora [11]. In this study, we employed WGS to conduct a comparative genomic analysis of environmental Lm, Li, Lw, and background microflora isolated from a dairy processing facility. Our objectives were to (i) identify and compare common genetic determinants associated with stress tolerance, biofilm formation, and persistence; (ii) explore the functional potential of environmental non-pathogenic spp. and other environmental microbiota in modulating Lm persistence. This integrative approach provides a broader framework for understanding microbial dynamics in food processing ecosystems and identifies potential microbial targets for environmental pathogen control.

2. Materials and Methods

2.1. Environmental Sample Collection from a Dairy Processing Plant

Environmental samples comprising air and floor swab samples were collected from a commercial dairy processing plant with fully automated production processes in the USA in December 2023. Sampling was conducted concurrently across four distinct production lines, each involved in manufacturing different ice cream varieties. Samples were collected during production from multiple non-food-contact surfaces along the processing lines, such as under the freezers, close to flavor tanks, around drains on the floor, and near the ingredient dispensing units.
Floor swab samples were collected from five adjacent locations at each production line using EZ Reach™ sponge samplers (World Bioproducts©, Bothell, WA, USA), as described in our previous study [17]. Airborne cultivable microorganisms at each production line were sampled from four directional quadrants (north, south, east, and west) using Agar Strips Total Count (HYCON®, Merck KGaA, Darmstadt, Germany), as described in our previous study [17]. In total, 20 floor swab samples (5 locations per production line) and 16 air samples (4 locations per production line) were collected and analyzed. The sponges and agar strip samples were stored under refrigerated conditions and shipped overnight to the laboratory.

2.2. Isolation and Identification of Environmental Microflora

Swab samples were processed as described in our previous study [17], including serial dilutions in phosphate-buffered saline (PBS, pH 7.2) (Fisher Scientific, Waltham, MA, USA) and spread-plating on Tryptic Soy agar (TSA) (RemelTM, Thermo Fisher Scientific, Waltham, MA, USA). After incubation at 37 °C for 24–48 h, morphologically distinct colonies were Gram-stained and identified using Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry.
The agar strips of air samples were incubated at 37 °C for 24–48 h to allow visible colony development. Morphologically distinct colonies were then Gram-stained and identified via MALDI-TOF mass spectrometry. Each resulting culture from swab and air samples was treated as a representative environmental culture for downstream identification and sequencing.

2.3. Sourcing of Listeria Isolates

This study utilized six previously characterized environmental isolates of Listeria spp., obtained from non-food-contact surfaces (floors and drains) in commercial dairy manufacturing facilities. The isolates included three Li (634-25, 634-34-S-5, and 634-34-S-6), two Lw (634-3 and 634-253-S-5), and one Lm (315-S1). All had been previously characterized by ribotyping. Additionally, four American Type Culture Collection (ATCC) strains were used as reference controls: L. welshimeri ATCC 35897, L. innocua ATCC 33090 and BAA 680 and, and Lm ATCC 51414. Glycerol stocks of environmental and Listeria isolates, as well as ATCC strains, were prepared as previously described in our study [17].

2.4. DNA Extraction from Environmental Cultures and Listeria Isolates

The activated cultures were centrifuged at 16,000× g for 2 min to obtain the cell pellet. The cell pellets were resuspended in 480 μL of 50 mM Ethylenediaminetetraacetic acid (EDTA) (pH 8; Thermo Fisher Scientific, Waltham, MA, USA) and incubated with 60 μL of Lysozyme (50 mg/mL, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 60 min. The genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA). DNA quantity and quality were evaluated using the Qubit™ dsDNA HS Assay Kit (Q32851) with a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

2.5. Library Preparation and Whole-Genome Sequencing (WGS) of Listeria Isolates: Oxford Nanopore Corrected Assembly with Illumina Reads

The library preparation and sequencing were done in the SDSU Genomics Sequencing Facility (South Dakota State University, Brookings, SD, USA). DNA libraries for MiSeq sequencing were prepared using the Illumina Nextera XT or Nextera Flex Library Preparation Kit (Illumina, San Diego, CA, USA) and sequenced on the Illumina MiSeq® platform (2 × 150 bp) (Illumina, San Diego, CA, USA) using the MiSeq Reagents V2 300 cycles kit (Illumina, San Diego, CA, USA). Raw reads were quality-controlled using CLC Genomics Workbench version 12.0.3 (Qiagen, Venlo, Netherlands), and reads with an average Phred quality score < 20 were discarded; no additional trimming parameters were applied. De novo assemblies were constructed using a length fraction of 0.5, a similarity fraction of 0.8, and a minimum contig length of 3000 bp.
In addition, long-read sequencing was performed on Listeria isolates using the Oxford Nanopore MinION® platform. Libraries were prepared using the Ligation Sequencing Kit v9 (SQK-LSK109) (Oxford Nanopore Technologies, Oxford, UK) and sequenced on an R9.4.1 flow cell for 72 h. Base calling was performed with Guppy version 4.4.2, followed by assembly using Flye version 2.8.3, and polishing with Nanopolish version 0.13.2 and Racon version 1.4.3.
Assemblies from Nanopore long-reads were corrected using the Illumina short-reads using the ‘Polish with Reads’ tool in the Long Read Support module of CLC Genomics Workbench vs. 23.0., which uses Racon [18]. A partial order alignment (POA) window of 500 bp was used in this step. Genomes were annotated using the RAST server (RASTtk pipeline)with default annotation parameters [19]. The stress tolerance, biofilm formation, and other persistence-related subsystems were identified using a Basic Local Alignment Search Tool (BLAST)-based approach [20].
FASTA files of the genome assemblies were uploaded to the Listeria multi-locus sequence typing (MLST) database (BIGSdb-Lm: http://bigsdb.pasteur.fr/listeria) (Accessed on 23 July 2025) hosted by the Pasteur Institute, France, for allele ID assignment [21]. Based on the seven housekeeping genes, MLST profiles including sequence type (ST) and sub-lineage (SL) were generated for each ATCC and environmental Listeria isolate using the BIGSdb-Pasteur platform, with the Listeria database submitted at the Pasteur Institute (IDs: 101844–101853).

2.6. Library Preparation, WGS, and Analysis of Environmental Microflora

DNA libraries were prepared using the Rapid Barcoding Kit 24 (SQK-RBK114.24) with 50 ng DNA per sample and sequenced on a PromethION sequencer (Oxford Nanopore Technologies, Oxford, UK). Assemblies were generated using Flye v2.8.3 and polished using Nanopolish v0.13.2, followed by Racon v1.4.3. Annotation was performed using RAST. Taxonomic identification was conducted by comparing 16S rRNA sequences obtained from the assembled genomes against the NCBI database using BLAST+v. 2.16.0+ [22]. All sequences were deposited in the NCBI database under accession numbers SAMN53082930–SAMN53082951, associated with BioProject PRJNA1357355.

2.7. Functional Annotation and Subsystem Analysis Using RAST

Annotated genomic data from RAST were organized into four levels: category, subcategory, subsystem, and role. To explore functional differences in Listeria isolates and environmental cultures, two separate visualizations were generated using JMP® software Student Version 18 (Cary, NC, USA). The first visualization compared subsystems present among all samples while also displaying associated categories and subcategories. The second focused on the “role category”. By utilizing the “role” data assigned by RAST, we compared specific protein functions across Listeria isolates and environmental samples (air and floor swabs). RAST identifies protein-encoding genes and then assigns them to specific functional roles based on manually curated subsystems. These functional roles represent the functions of the proteins themselves, not the genes directly [19]. This level of analysis allowed us to explore and compare functional variations at the protein level, revealing genes potentially linked to biofilm formation and persistence mechanisms among the Listeria isolates and environmental microflora.

3. Results and Discussion

3.1. Listeria Isolates Typing

The genome assemblies of all environmental Listeria isolates ranged from 2820 to 3166 kb, consistent with previously reported genome sizes for Lm (Table S1) [23]. MLST sequence tools are used to characterize sequence types (STs) and assess closely related sequences that may persist in FPEs [10]. The reference strain Lm ATCC 51414, isolated from raw milk associated with a listeriosis outbreak, was identified as ST 51414. In contrast, the processing environment isolate Lm 315-S-1 was classified as ST 5, indicating distinct clonal lineages [24] (Table 1). ST 5 has been repeatedly recovered from FPEs, highlighting its adaptation to industrial niches and resilience to routine sanitation procedures, distinguishing it from transient strains [25]. MLST revealed heterogeneity among all isolates except Lw 634-3 and Lw 634-253-S-5, which shared sequence type (ST-2688), indicating clonal relatedness and suggesting a common origin [24].

3.2. Characterization and Comparison of the Microflora from Environmental Culture

A total of 22 environmental cultures were obtained and sequenced from non-food-contact surfaces of four production lines in the dairy processing facility. Of these, 12 cultures were recovered from surface swab samples (designated S1–S12), and 10 were obtained from air samples (A1–A10). All the environmental cultures were first identified using MALDI-TOF mass spectrometry. Species-level identifications obtained from 16S rRNA identification were used to compare and characterize the environmental microflora (Table 2). Although MALDI-TOF analysis typically yielded a single taxonomic identification per colony, WGS revealed the presence of multiple taxa within the same culture. These findings suggest that some colonies may have consisted of mixed microbial populations despite appearing morphologically uniform. They are thus referred to as environmental cultures rather than pure isolates in this study. The environmental cultures exhibited a much broader genome size range (2237 to 18,613 kb), reflecting considerable taxonomic and functional diversity (Table S1).
Stenotrophomonas was identified as the predominant species in the air samples and has been previously isolated from the dairy processing environment [26]. In contrast, the floor swab samples revealed a distinct microbial profile, with Pseudomonas spp., Raoultella spp., and Morganella spp. as the predominant taxa. Pseudomonas is among the most reported genera in FPEs, particularly after sanitation procedures [27]. Its persistence is closely linked to its high stress tolerance as well as its ability to form biofilms [28,29]. No genera were common to both the floor swab and the air sample. The limited overlap in microflora indicates that aerosolization from floor surfaces and drains within the processing area was likely minimal.

3.3. Characterization and Comparison of the Subsystems Identified from Environmental Cultures and Listeria Isolates

The categories selected for this study were derived from the RAST annotation output and specifically chosen to examine persistence-associated subsystems, with a particular focus on biofilm formation and stress response. Accordingly, the analysis was limited to the following seven subsystems: cell wall and capsule, membrane transport, motility and chemotaxis, regulation and cell signaling, stress response, virulence, disease and defense, and dormancy and sporulation.
The category, subcategory, and subsystems are illustrated in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6 using a heat map, where each column represents an isolate/culture, and each row corresponds to a specific subsystem. Colored cells indicate the presence and abundance of subsystem-associated genes, with darker red representing higher gene counts and darker blue indicating lower counts. Blank or white cells denote the absence of genes within a given subsystem.

3.3.1. Cell Wall and Capsule Category

The cell wall and capsule are crucial to bacterial persistence, supporting cellular integrity, adhesion, and resistance to environmental stressors essential for long-term survival and biofilm formation in harsh environments [30,31,32]. In our analysis, genes associated with Gram-positive cell wall components were more abundant in Listeria isolates than in air and floor swab samples, consistent with their Gram-positive classification (Figure 1).
We identified nine capsular and extracellular polysaccharide subsystems, known to contribute to biofilm formation. Capsular polysaccharides regulate biofilm size, adhesion, aggregation, and stress resistance [30]. CMP-N-acetylneuraminate and sialic acid pathways, dTDP-rhamnose genes, and EPS biosynthesis support adhesion, matrix formation, and persistence, with deacetylases enhancing adhesiveness [31,33,34,35]. The Streptococcal hyaluronic acid capsule similarly promotes adhesion and biofilm structure [36]. Our comparative analysis revealed that genes associated with this category were more prevalent in floor swab cultures, followed by air cultures, and were least abundant in Listeria isolates, indicating their role in forming stable biofilm communities for long-term survival [37]. Notably, the Lm isolate harbored more capsular and polysaccharide-associated genes than Lw and Li isolates, suggesting greater biofilm-forming ability; however, this requires further validation due to the limited number of isolates. The complete absence of these genes in the Li isolates highlights species-specific variability in environmental adaptation (Figure 1). Furthermore, comparative analysis revealed that subsystems within the “cell wall and capsule, no sub-category,” including peptidoglycan recycling genes, were more prevalent in all Listeria isolates, followed by air, and least abundant in floor swabs. These genes have been implicated in survival under stress conditions such as high temperatures, low pH, and nutrient limitation [32], indicating their potential role in supporting Listeria persistence in dairy processing environments.

3.3.2. Membrane Transport Category

We examined this category due to its role in mediating bacterial interactions with the environment, which influence adhesion and biofilm formation [38]. The main subsystems identified in our study related to biofilm formation were the protein secretion systems, including Type I–V, VII, and VIII, previously reported to contribute to biofilm development by exporting proteins that function as adhesins and promote matrix formation [38,39,40]. Genes corresponding to these systems were most prevalent in floor swab cultures, followed by air cultures, and were absent in Listeria isolates (Figure 2), suggesting that environmental microflora utilize diverse secretion systems to establish and maintain biofilms, whereas Listeria spp. may rely on alternative adhesion mechanisms. Additionally, genes encoding bacterial ABC transporters implicated in adhesion and biofilm formation [41] were detected only in three floor swab cultures and the Lm ATCC 51414 strain, indicating a potential role in biofilm development for select strains.

3.3.3. Motility and Chemotaxis Category and Dormancy and Sporulation Category

The motility and chemotaxis categories were used to identify subsystems potentially involved in surface colonization and biofilm formation. Genes associated with flagellar motility and structure were absent in Listeria isolates, while Campylobacter-specific flagellar systems were more prevalent (Figure 3), possibly reflecting sequence or functional similarities [42]. In contrast, flagellar motility subsystems were detected in floor swabs and air cultures, supporting early biofilm formation by promoting surface movement, attachment, microcolony development, and colonization under FPE stress conditions [43].
Subsystems associated with dormancy and sporulation were more prevalent in air cultures than in floor swabs and Listeria isolates (Figure 3), reflecting the stress-prone nature of air, where spore formation enhances bacterial survival until conditions become favorable [44].

3.3.4. Regulation and Cell Signaling Category

Several subsystems associated with virulence regulation, toxin-antitoxin systems, quorum sensing, and biofilm formation were identified, highlighting their potential roles in environmental persistence. The CytR regulatory gene was detected in six floor swabs and one air culture but was absent in all Listeria isolates (Figure 4). CytR function is species-dependent: in Vibrio cholerae, it represses the vps gene cluster, inhibiting EPS production and biofilm formation [45], whereas in Pectobacterium carotovorum, it promotes biofilm formation [46]. Its presence in environmental cultures, but not in Listeria, suggests distinct regulatory strategies among taxa in the same FPEs.
A biofilm-adhesion biosynthesis subsystem, including PgaA–D proteins, was found in five air and three floor swab cultures. The pgaABCD operon encodes a key polysaccharide adhesin essential for biofilm formation [47], indicating enhanced adhesion capacity and potential for biofilm establishment on FPE surfaces.
Toxin-antitoxin systems, including Phd-Doc, YdcE-YdcD, and MazEF, were present in all Listeria isolates and a few environmental cultures. These systems prevent cell death or growth inhibition and support survival under nutrient limitation, antibiotics, and oxidative stress [48,49], suggesting a persistence strategy that enables Listeria to withstand fluctuating FPE conditions despite routine sanitation.
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Figure 1. Distribution of subsystems classified under the cell wall and capsule category.
Figure 1. Distribution of subsystems classified under the cell wall and capsule category.
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Figure 2. Distribution of subsystems classified under the membrane transport category.
Figure 2. Distribution of subsystems classified under the membrane transport category.
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Figure 3. Distribution of subsystems classified under the categories: Dormancy and Sporulation, and Motility and Chemotaxis.
Figure 3. Distribution of subsystems classified under the categories: Dormancy and Sporulation, and Motility and Chemotaxis.
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Figure 4. Distribution of subsystems classified under the regulation and cell signaling category.
Figure 4. Distribution of subsystems classified under the regulation and cell signaling category.
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3.3.5. Stress Response Category

Subsystems related to detoxification and osmotic stress were identified across all sample types, with higher prevalence in floor swabs cultures. Genes associated with oxidative stress, carbon starvation, and the universal stress protein family were more abundant in floor swabs and air cultures compared to Listeria isolates, suggesting broader stress adaptation in environmental microflora (Figure 5). Oxidative stress-related genes were the most prominent across all isolates/cultures, underscoring their essential role in protecting bacteria against reactive oxygen species and other environmental challenges [50].
Genes involved in the periplasmic stress response were present in all Listeria isolates and swab cultures, and most air cultures, with the highest prevalence observed in swab cultures. This response system maintains cell envelope integrity under heat, pH fluctuations, and antimicrobial exposure [51]. These results collectively suggest that environmental cultures, particularly from floor swabs, exhibit a broader and more diverse stress-response than Listeria isolates, highlighting the importance of supporting bacterial survival and persistence in the harsh conditions of the FPEs.

3.3.6. Virulence and Disease Category

Subsystems associated with resistance to antibiotics and toxic compounds were identified. These included mechanisms against fluoroquinolones, streptothricin, tetracycline, fosfomycin, β-lactams, and multidrug resistance (MDR) efflux pumps, and tolerance to heavy metals such as copper, cadmium, mercury, and chromium. MDR efflux pumps, including the MexC-MexD-OprJ system, actively extrude antimicrobials, toxins, and environmental stressors, enhancing bacterial survival under harsh FPE conditions [52,53]. In our study, MDR efflux pumps were most prevalent in Listeria isolates, followed by floor swabs and air samples (Figure 6), indicating an enhanced ability to survive antimicrobial interventions and potentially persist in FPEs. Several floor swabs and air cultures also harbored resistance determinants, suggesting that environmental microbiota serve as reservoirs of resistance traits. Repeated exposure to disinfectants and residual antimicrobials may allow these strains to accumulate resistance genes, potentially facilitating horizontal gene transfer to Listeria or other foodborne pathogens [54]. These findings highlight the importance of monitoring both pathogenic isolates and the surrounding microbial community, as it may influence persistence and resistance dynamics in the processing environment.
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Figure 5. Distribution of subsystems classified under the stress response category.
Figure 5. Distribution of subsystems classified under the stress response category.
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Figure 6. Distribution of subsystems classified under the virulence and disease category.
Figure 6. Distribution of subsystems classified under the virulence and disease category.
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3.3.7. Genomic Comparison of Subsystems in Listeria and Environmental Cultures

In the studied subsystems, Listeria isolates exhibit genomic potential to survive under adverse conditions, with key traits related to biofilm formation, stress resistance, and antimicrobial resistance. Collectively, these genomic features indicate that Lm, Li, and Lw possess the intrinsic capacity to independently initiate and maintain biofilm, supporting their survival and persistence in FPEs. Compared to environmental cultures (air and swab), Listeria isolates possessed fewer genes associated with biofilm development, reflecting species-specific adaptations that favor survival under selective pressures such as sanitation and temperature fluctuations. In contrast, environmental cultures demonstrate a broader representation of genes related to biofilm formation and stress response, suggesting greater capacity to establish stable biofilm communities on surfaces.
While Lm can establish monospecies biofilms, its persistence in multispecies environments likely relies on opportunistic integration rather than competitive dominance, exploiting extracellular polymeric substances produced by other microbes [55]. The capacity to integrate and persist in multispecies biofilms complicates eradication efforts in FPEs [56] and highlights the need to consider the entire microbial community, rather than pathogens alone, in strategies to prevent contamination and persistence.

3.3.8. Comparing Genomic Determinants of Persistence Among Listeria Species

Comparison of the three Listeria species, Lm, Lw, and Li, revealed species-level differences in genetic subsystems associated with biofilm formation. Although many subsystem categories were shared, Lm isolate exhibited more genes related to capsular and extracellular polysaccharide production, supporting stronger biofilm structure and adhesion. In contrast, Li lacked these genes, and Lw had fewer than Lm, suggesting a gradient in genetic potential for biofilm: Lm > Lw > Li. This profile aligns with previous findings that Lm adheres more effectively to surfaces in pure cultures compared to Li, reinforcing the idea that Lm is more genetically equipped for biofilm initiation. However, our results differ from other studies showing that Li can outcompete Lm in mixed-species biofilms [57], and its stronger adhesion at lower temperatures (e.g., 12 °C) and a faster growth rate may explain its higher prevalence in FPEs [58]. Li may possess additional genetic determinants not studied in this study, contributing to competitive advantage in multispecies biofilms. Given the limited number of isolates analyzed, a broader strain set could provide deeper insight into the species-specific biofilm mechanisms.
Most other subsystem categories were comparable across species, though some specific differences were observed; for example, ABC transporter genes associated with adhesion and matrix development were present only in the Lm ATCC strain. Overall, these findings highlight species-level variability in biofilm-related genetic determinants and underscore the need for broader genomic surveillance to understand the Listeria persistence strategies in FPEs.

3.4. Characterization and Comparison of Functional Roles Assigned to Listeria Isolates and Environmental Cultures

As outlined in the Section 2, the second layer of our genomic analysis focused on the RAST “role category”, which classifies protein-encoding genes into functional roles based on subsystems. By analyzing these role categories, we identified distinct patterns in persistence potential, particularly in functions associated with biofilm formation and stress response. A total of 100 genes were analyzed across all Listeria isolates and environmental cultures, which were categorized based on their function into categories: flagellar assembly and motility, biofilm, stress response, DNA repair and recombination, and SigB operon.

3.4.1. Flagellar Assembly and Motility Genes in Listeria Isolates and Environmental Cultures

A total of 47 protein-coding genes associated with flagellar assembly and motility were identified across the dataset (Figure 7). FlaA, encoding flagellin A, a key structural component essential for motility and initial surface attachment [59], was present in all Listeria isolates and a subset of swab and air culture. Other core flagellar proteins detected in the Listeria included FlgB, FlgC, and FliE, which are involved in basal body and hook assembly as part of the flagellar structure [8,60]. In contrast, swab and air cultures exhibited a broader suite of motility-associated proteins, including DegQ, DegS, FlaB, FlbB, FlbD, FleN, FleQ, FleS, FlgA, FlgD–N, FlhA–F, FliF–T, and MotA, MotB, and MotY, which contributes to flagellar biosynthesis, structural assembly, regulation, and motor function [60,61,62,63,64]. Air cultures (A4, A6, A7, and A8) and several swab cultures (S5, S6, S7, S11, S12) harbored a significantly higher number of these genes compared to Listeria isolates, whereas some swabs and air cultures (A1, A2, S9) lacked all studied genes. This absence in certain environmental cultures is surprising, given their surface association and biofilm potential, and may reflect reliance on the collective motility of surrounding microbes or alternative colonization mechanisms, such as type IV pili or EPS mediated adherence [65]. Overall, Listeria isolates exhibited fewer flagellar genes compared to environmental (swab and air) cultures.

3.4.2. Biofilm-Associated Genes in Listeria Isolates and Environmental Cultures

A total of 11 protein-coding genes associated with biofilm formation were identified (Figure 8). ActA and PrfA were exclusively detected in the Lm isolate and Lm ATCC culture. ActA encodes the actin assembly-inducing protein, promoting bacterial aggregation and biofilm development [59], while PrfA functions as a master transcriptional regulator controlling virulence genes, including ActA, and contributes to later stages of biofilm maturation [59]. Their presence solely in the Lm strain underscores their importance in pathogenicity and biofilm robustness, likely enhancing persistence in FPEs.
The Slr protein, encoded by the SlrR and SlrA genes, was identified in two air sample cultures (A4 and A6), both taxonomically classified as Bacillus spp. (Table 2). This protein regulates biofilm formation while repressing flagellar motility, enabling a switch between motile and sessile states [66]. These cultures also harbored the regulatory gene abrA, which negatively regulates biofilm initiation and Slr expression, suggesting a tight control of early biofilm development [67]. EPS biosynthesis genes EpsC and EpsD were detected in air and floor swab cultures, contributing to surface adhesion, stress protection, and nutrient acquisition [68].
The PhoPR two-component system, present in multiple air and floor swab cultures and absent in Listeria isolates, has been linked to phosphate sensing and enhanced biofilm formation under nutrient-limited conditions [10]. Additionally, DltB and DltD, detected more in Listeria isolates, modify teichoic acids to affect surface charge and adhesion, and with deletion reducing biofilm formation [10]. The LuxR protein was found in three floor swab cultures, and as a key regulator of quorum sensing and EPS production, may influence biofilm matrix and microbial community behavior [69].
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Figure 7. Protein-encoding genes related to flagellar assembly and motility in the environmental cultures and Listeria isolates.
Figure 7. Protein-encoding genes related to flagellar assembly and motility in the environmental cultures and Listeria isolates.
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Figure 8. Protein-encoding genes related to stress response, SigB operon, DNA repair and recombination, and biofilm formation in the environmental cultures and Listeria isolates.
Figure 8. Protein-encoding genes related to stress response, SigB operon, DNA repair and recombination, and biofilm formation in the environmental cultures and Listeria isolates.
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Collectively, these results reveal distinct biofilm-related genetic profiles between Listeria and background environmental cultures. While Lm carries biofilm-associated genes like ActA and PrfA, environmental microbes contribute structural and regulatory elements such as EpsC, EpsD, Slr, and LuxR. These genetic traits may support the formation of resilient, multispecies biofilms that protect Listeria in dairy processing environments.

3.4.3. Stress Response Genes: The SigB Operon and Alternative Sigma Factors

A total of 10 protein-coding genes associated with the SigB operon were identified across the isolates and cultures (Figure 8). The SigB operon mediates adaptation to environmental stress, activating general stress response genes, supporting biofilm formation in Lm, and enhancing resistance against disinfectants [10]. SigH, RsbS, RsbT, RsbU, and RsbW were detected in all Listeria isolates, variably in air samples, and least in swab samples, indicating species- and environment-specific distribution. SigH acts as a global regulator under heat shock, oxidative stress, and cell-wall damage [70], while the Rsb pathway (RsbS/RsbT stress-sensing complex and RsbU/RsbW/RsbV phosphorylation-based signal transduction) governs SigB activation [71].
Four alternative sigma factors, RpoE, RpoH, and RpoN, were examined for their known roles in environmental stress responses. RpoE and RpoH mediate envelope and cytoplasmic heat shock proteins [72]. RpoE and RpoH were absent in Listeria isolates but present in a few environmental cultures, suggesting differential regulatory strategies between Listeria and the background microbiota. RpoN, detected in Listeria, regulates motility, stress resistance, and biofilm formation [73], highlighting its role in adaptation and persistence in hostile environments such as dairy processing environments.
Overall, the enhanced presence of SigB operon genes and RpoN in Listeria isolates reflects their intrinsic stress tolerance, whereas the exclusive occurrence of RpoE and RpoH in environmental cultures suggests that the broader microbiota may deploy distinct and complementary mechanisms to endure stress, likely influencing multispecies interactions and survival under FPEs.

3.4.4. DNA Repair and Recombination Genes in Listeria Isolates and Environmental Cultures

Nine protein-coding genes (RecA, RecN, RecO, RecQ, RecR, RecS, RecU, RecF, and RecX) were analyzed (Figure 8), as they are critical for bacterial survival under genotoxic stress (DNA-damaging stress) typical of FPEs [74]. All Listeria isolates harbored the complete set of these genes, underscoring their capacity to withstand oxidative damage, disinfectants, UV exposure, and desiccation, conditions commonly encountered in dairy processing settings [75]. RecA was highly abundant across all isolates and cultures and as a key mediator of homologous recombination and the central regulator of the SOS response (response to significant DNA damage), facilitating DNA repair following damage [76]. Its high abundance in all samples suggests a strong selective pressure for maintaining DNA repair competency in both Listeria and environmental cultures.
Most swab and air cultures also encoded these genes, although with greater variability than observed in Listeria isolates, suggesting that while DNA repair is broadly conserved, gene representation may differ by ecological niche and exposure history. Overall, the widespread presence of DNA repair and recombination genes underscores the genetic resilience of microbial communities in dairy processing environments.

3.4.5. Stress Response Genes in Listeria and Environmental Isolates

A total of 22 protein-coding genes associated with stress tolerance and survival mechanisms were identified across the isolates and cultures (Figure 8). The Bcr family proteins, which confer resistance to benzalkonium chloride (disinfectant), were found in all Listeria isolates, followed by swab and air culture, supporting earlier findings that disinfectant use may exert strong selective pressure, driving adaptation and persistence [10]. Among acid tolerance genes, AdiA (arginine decarboxylase) was detected exclusively in air culture, CadB (lysine/cadaverine antiporter) was found in both air and swab culture [77], and ArcC (Carbamate kinase), which has been reported to support pH regulation under acidic stress, was identified in Lm, air, and swab culture [78]. These acid response genes were more varied among environmental cultures than in Listeria, reflecting niche-specific acid adaptation.
Heat shock and protein maintenance systems were also represented. GrpE, DnaJ, and DnaK were consistently present in all isolates and cultures, reflecting their essential roles in protein folding and nucleic acid protection during stress [79,80]. CtsR, together with ClpA, ClpS, and ClpX, was detected only in air and swab isolates, indicating environment-specific regulation of class III heat shock genes [81]. In contrast, ClpB, a Class III heat shock protein, a key protein disaggregation factor under extreme conditions [82], was exclusive to the Lm isolate, suggesting stress-specific specialization in pathogenic strains. The Opu osmoprotectant uptake system, comprising OpuAA, OpuAB, OpuAC, OpuBA, OpuBB, OpuBC, OpuBD, and OpuD, was present in all Listeria isolates, followed by air, and least in swab cultures, consistent with its established role in osmoregulation and thermal stress survival [83].
Comparatively, Listeria isolates contained a more consistent core of stress-related genes, particularly for disinfectant and osmotic stress resistance, whereas air and swab cultures displayed greater diversity in acid and heat shock-related genes. This likely reflects adaptation to different environmental pressures within FPEs, where microbial diversity contributes to resilience and persistence.

3.4.6. Comparison and Contextual Considerations

In summary, the comparative genomic analysis reveals that environmental Listeria isolates, including Lm, have a genetic ability for biofilm formation, stress tolerance, and DNA repair. In contrast, other environmental cultures (from air and swab samples) show a broader range of genetic potential, including motility, biofilm regulation, and specific stress adaptations, suggesting that these microbes could contribute to multispecies biofilms that can provide a protective environment for Lm in FPEs. Overall, the interactions between environmental Lm and surrounding microbes can play a key role in its persistence under food processing conditions. Studying the genes related to persistence can thus help develop better control strategies, improve detection methods for injured Listeria cells, and explore biocontrol approaches, ultimately enhancing food safety and regulatory measures in FPEs.
Despite these insights, a few limitations should be considered when interpreting the results. The study utilized a limited number of environmental Listeria isolates, which does not fully capture the genomic diversity of environmental Listeria spp. from dairy processing environments. Moreover, while genomic analysis provides insight into genetic potential, RNA sequencing would yield a more accurate picture of active metabolic and stress response pathways [84], especially under real-time processing conditions.
Future studies may thus incorporate larger, more diverse collections of isolates from multiple dairy facilities and processing contexts. Integrating genome-wide association studies with transcriptomic and phenotypic data will enhance the robustness of genotype-to-function inferences and improve our understanding of microbial persistence in food production environments.

4. Conclusions

The genomic analysis indicates that all examined Listeria species, including L. monocytogenes, L. welshimeri, and L. innocua, possess a shared set of stress response genes, biofilm-associated mechanisms, and DNA repair pathways, enabling each species to form biofilms independently and persist within dairy and food processing environments. Environmental cultures from air and floor swabs exhibit greater genetic diversity in stress adaptation and biofilm-associated genes, highlighting the potential for multispecies biofilm communities that may enhance L. monocytogenes survival within these environments. These findings underscore the importance of considering microbial community dynamics, rather than individual pathogens alone, when developing sanitation and contamination prevention strategies for food processing environments.
Future transcriptomic studies may provide deeper insight into how environmental Listeria and background microbiota regulate persistence-associated genes under food processing conditions. Additionally, exploring the relationship between biofilm-forming capacity and persistence-related genetic subsystems may further improve our understanding of L. monocytogenes survival and inform effective microbial control strategies in food production environments.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/applmicrobiol6010020/s1, Table S1: Genome characteristics of environmental cultures and Listeria isolates.

Author Contributions

Conceptualization, V.P., S.A., J.L.G.-H. and B.K.; methodology, V.P., S.A. and J.L.G.-H.; data curation, V.P.; writing—original draft preparation, V.P.; writing—review and editing, V.P., S.A., J.L.G.-H. and B.K.; visualization, S.A.; supervision, S.A.; project administration, S.A.; funding acquisition, S.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Dairy Management Inc. (Rosemont, IL, USA), grant number 3X4105.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors would like to acknowledge the funding support from Dairy Management Inc., as well as the infrastructure support from the Department of Dairy and Food Science, SDSU, and the Agricultural Experimental Station, SDSU. The genomic sequencing work was conducted using the South Dakota State University Genomics Sequencing Facility (RRID:SCR_023959) and the Functional Genomics Core Facility (RRID:SCR_023786) supported in part by the National Science Foundation/EPSCoR Grant No. 0091948, the South Dakota Agricultural Experiment Station, and by the State of South Dakota. All authors have consented to the acknowledgment.

Conflicts of Interest

Author Brian Kraus is employed by the Wells Enterprises Inc. The remaining authors declare that the research reported in this article was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
LmListeria monocytogenes
FPEsFood processing environments
EPSExtracellular polymeric substances
MALDI-TOFMatrix-assisted laser desorption ionization–time of flight
LwListeria welshimeri
LiListeria innocua
MLSTMulti-locus sequence typing
WGSWhole-genome sequencing
RASTRapid annotations utilizing subsystems technology

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Table 1. Multi-locus sequence typing results of the Listeria isolates based on the corrected assembly.
Table 1. Multi-locus sequence typing results of the Listeria isolates based on the corrected assembly.
Listeria IsolatePasteur IDClonal Complex (CC)Sublineage
(SL)		Phylogenetic LineagecgMLST TypeSequence Type (ST)Novel Alleles
Li 634-25101847CC1008N.A.L. innocuaN.A.ST313768
Li 634-34-S-5101848CC1008SL1008L. innocuaN.A.ST100865
Li 634-34-S-6101849CC14891489L. innocuaN.A.ST1489200
Li ATCC 33090101852ST139SL139L. innocuaN.A.ST139155
Li ATCC BAA 680101851CC140SL140L. innocuaN.A.ST14078
Lm 315-S-1101844CC5SL5IN.A.ST519
Lm ATCC 51414101853CC4SL5ICT13172ST558
Lw 634-3101845ST26882688L. welshimeriN.A.ST2688476
Lw 634-253-S-5101846ST2688SL2688L. welshimeriCT13173ST2688467
Lw ATCC 35897101850CC129SL129L. welshimeriN.A.ST129116
Table 2. Identification of environmental cultures from swab and air samples using the 16S rRNA identification and MALDI-TOF MS.
Table 2. Identification of environmental cultures from swab and air samples using the 16S rRNA identification and MALDI-TOF MS.
Sample TypeSample ID16S rRNA IdentificationMALDI-TOF MS
Greatest Identity %Identity
AirA197.893–99.933Staphylococcus pasteuriStaphylococcus pasteuri
A299.858–99.929Micrococcus aloeveraeMicrococcus luteus
A399.352–99.545Stenotrophomonas maltophiliaStenotrophomonas maltophilia
99.35Stenotrophomonas forensis
A499.74–99.935Bacillus halotoleransBacillus sp.
99.458Stenotrophomonas lactitubi
99.584–99.722Stenotrophomonas cyclobalanopsidis
A699.722Stenotrophomonas cyclobalanopsidisStenotrophomonas maltophilia
99.74–99.935Bacillus halotolerans
A799.796–100Rummeliibacillus stabekisiiStaphylococcus epidermidis
A899.73Paenibacillus glucanolyticusRummeliibacillus stabekisii
99.257–100Rummeliibacillus stabekisii
A999.19–99.932Paenibacillus glucanolyticusPaenibacillus glucanolyticus
A1099.655–99.793Pantoea agglomeransStaphylococcus warneri
SwabS199.993–100Pseudomonas aeruginosaPseudomonas aeruginosa
S299.33–100Ectopseudomonas oleovoransPseudomonas oleovorans
S399.25–99.386Pseudomonas oryzihabitansRaoultella ornithinolytica
99.725–99.862Raoultella terrigena
S499.793–99.862Lelliottia amnigenaCitrobacter gillenii
99.672–99.803Citrobacter gillenii
99.503–99.858Citrobacter arsenatis
S599.725–99.863Pseudomonas koreensisPseudomonas koreensis
S699.48–99.87Aeromonas hydrophilaRaoultella ornithinolytica
99.558Morganella morganii subsp. sibonii
99.582Raoultella ornithinolytica
99.851Klebsiella grimontii
99.714–99.93Raoultella planticola
99.787Citrobacter arsenatis
100Huaxiibacter chinensis
S799.8–99.933Lactococcus lactisRaoultella planticola
99.798–99.865Enterococcus gallinarum
99.41–99.705Morganella morganii subsp. sibonii
98.498–98.567Providencia heimbachae
98.372–98.641Providencia burhodogranariea
S899.591–99.659Serratia marcescensSerratia marcescens
99.41–99.705Morganella morganii subsp. sibonii
99.933Lactococcus lactis
99.786–100Raoultella planticola
S9100Leuconostoc mesenteroidesLeuconostoc mesenteroides
S1099.589–99.795Acinetobacter lwoffiiRahnella aquatilis
99.717–99.788Prolinoborus fasciculus
S1199.391–99.661Rahnella inusitataExiguobacterium mexicanum
99.914Exiguobacterium artemiae
100Leuconostoc mesenteroides
S1299.218–99.87Shewanella xiamenensisShewanella oneidensis
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Poswal, V.; Anand, S.; Gonzalez-Hernandez, J.L.; Kraus, B. Genetic Determinants Associated with Persistence of Listeria Species and Background Microflora from a Dairy Processing Environment. Appl. Microbiol. 2026, 6, 20. https://doi.org/10.3390/applmicrobiol6010020

AMA Style

Poswal V, Anand S, Gonzalez-Hernandez JL, Kraus B. Genetic Determinants Associated with Persistence of Listeria Species and Background Microflora from a Dairy Processing Environment. Applied Microbiology. 2026; 6(1):20. https://doi.org/10.3390/applmicrobiol6010020

Chicago/Turabian Style

Poswal, Vaishali, Sanjeev Anand, Jose L. Gonzalez-Hernandez, and Brian Kraus. 2026. "Genetic Determinants Associated with Persistence of Listeria Species and Background Microflora from a Dairy Processing Environment" Applied Microbiology 6, no. 1: 20. https://doi.org/10.3390/applmicrobiol6010020

APA Style

Poswal, V., Anand, S., Gonzalez-Hernandez, J. L., & Kraus, B. (2026). Genetic Determinants Associated with Persistence of Listeria Species and Background Microflora from a Dairy Processing Environment. Applied Microbiology, 6(1), 20. https://doi.org/10.3390/applmicrobiol6010020

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