Nonpathogenic Listeria Research Articles

Comparative genomic analysis of pathogenic factors of Listeria spp. using whole-genome sequencing

Listeria monocytogenes is an important foodborne pathogen known for causing listeriosis. To gain insights into the pathogenicity, genetic characterization, and evolution of various Listeria species, in vitro cell adhesion and invasion ability assays and whole-genome sequencing were performed using four Listeria strains isolated from livestock and poultry slaughterhouses. The four Listeria strains exhibited adhesion and invasion abilities in Caco-2 and RAW264.7 cells. Pathogenic Liv1-1 and Lm2-20 had higher adhesion ability, but non-pathogenic Lin4-99 was more invasive than Lm2-20 (p < 0.05). Genetic characterization revealed the presence of a single chromosome without plasmid across four strains with similar whole-genome sizes and G + C% content. Analysis of key pathogenic genes underscored the presence of multiple virulence genes among the four Listeria strains. In contrast, non-pathogenic Listeria lacked LIPI-1, LIPI-2, and LIPI-3 genes, which could possibly be the cause of their non-pathogenicity despite their in vitro cell adhesion and invasion abilities. Thus, genetic determinants of Listeria do not necessarily predict cell adhesion and/or invasive ability in vitro. This study presents a comprehensive comparative genome-wide analysis of four Listeria strains, offering invaluable insights into the pathogenesis of the Listeria genus.

Prevalence, Antimicrobial Resistance, and Characterization of Listeria Spp. Isolated from Various Sources in Ethiopia: A Comprehensive Review.

Listeriosis is an important foodborne zoonotic disease affecting humans and animals in Ethiopia. This review aims to synthesize the epidemiology, prevalence, distribution, and antimicrobial resistance of Listeria species in the country. The literature reveals a widespread occurrence of Listeria infection in humans, animals, and food products, with an average prevalence of 21.6% for Listeria species and 6.9% for L. monocytogenes. Three sequence types (STs) of L. monocytogenes (2, 145, and 18) and twelve STs of L. innocua (1489, 1619, 603, 537, 1010, 3186, 492, 3007, 1087, 474, 1008, and 637) were reported from milk and dairy products. Contamination rates ranged from 4.1% to 42.9% across livestock, dairy, slaughterhouses, and processing facilities, indicating faults in production practices. Sporadic human listeriosis outbreaks have occurred since 1967, causing meningitis, perinatal infections, and deaths, with recent studies showing L. monocytogenes isolation in up to 10.4% of febrile patients, confirming foodborne transmission. Non-pathogenic Listeria species were also common on farms and in facilities. Ovine listeriosis poses a threat to Ethiopia's sheep and goat industries, with over 40% seroprevalence in some herds. Comprehensive control measures across the food chain are needed to curb contamination and protect public health. Isolates from various foods show antibiotic resistance to first-line agents but susceptibility to others like gentamicin and cephalosporins. In conclusion, this review synthesizes evidence on Listeria distribution in Ethiopia's food system and disease burden, highlighting the need for improved food safety policies and awareness.

Tetracycline resistance in Listeria monocytogenes and L. innocua from wild black bears (Ursus americanus) in the United States is mediated by novel transposable elements.

Listeria monocytogenes causes severe foodborne illness and is the only human pathogen in the genus Listeria. Previous surveys of AMR in Listeria focused on clinical sources and food or food processing environments, with AMR in strains from wildlife and other natural ecosystems remaining under-explored. We analyzed 185 sequenced strains from wild black bears (Ursus americanus) from the United States, including 158 and 27 L. monocytogenes and L. innocua, respectively. Tetracycline resistance was the most prevalent resistance trait. In L. monocytogenes, it was encountered exclusively in serotype 4b strains with the novel Tn916-like element Tn916.1039. In contrast, three distinct, novel tetracycline resistance elements (Tn5801.UAM, Tn5801.551, and Tn6000.205) were identified in L. innocua. Interestingly, Tn5801.551 was identical to elements in L. monocytogenes from a major foodborne outbreak in the United States in 2011. The findings suggest the importance of wildlife and non-pathogenic Listeria species as reservoir for resistance elements in Listeria.

Cell-surface anchoring of Listeria adhesion protein on L. monocytogenes is fastened by internalin B for pathogenesis

Listeria adhesion protein (LAP) is a secreted acetaldehyde alcohol dehydrogenase (AdhE) that anchors to an unknown molecule on the Listeria monocytogenes (Lm) surface, which is critical for its intestinal epithelium crossing. In the present work, immunoprecipitation and mass spectrometry identify internalin B (InlB) as the primary ligand of LAP (KD ∼ 42nM). InlB-deleted and naturally InlB-deficient Lm strains show reduced LAP-InlB interaction and LAP-mediated pathology in the murine intestine and brain invasion. InlB-overexpressing non-pathogenic Listeria innocua also displays LAP-InlB interplay. In silico predictions reveal that a pocket region in the C-terminal domain of tetrameric LAP is the binding site for InlB. LAP variants containing mutations in negatively charged (E523S, E621S) amino acids in the C terminus confirm altered binding conformations and weaker affinity for InlB. InlB transforms the housekeeping enzyme, AdhE (LAP), into a moonlighting pathogenic factor by fastening on the cell surface.

Genetic Characterization of Listeria from Food of Non-Animal Origin Products and from Producing and Processing Companies in Bavaria, Germany

Reported cases of listeriosis from food of non-animal origin (FNAO) are increasing. In order to assess the risk of exposure to Listeria monocytogenes from FNAO, the genetic characterization of the pathogen in FNAO products and in primary production and processing plants needs to be investigated. For this, 123 samples of fresh and frozen soft fruit and 407 samples of 39 plants in Bavaria, Germany that produce and process FNAO were investigated for Listeria contamination. As a result, 64 Listeria spp. isolates were detected using ISO 11290-1:2017. Environmental swabs and water and food samples were investigated. L. seeligeri (36/64, 56.25%) was the most frequently identified species, followed by L. monocytogenes (8/64, 12.50%), L. innocua (8/64, 12.50%), L. ivanovii (6/64, 9.38%), L. newyorkensis (5/64, 7.81%), and L. grayi (1/64, 1.56%). Those isolates were subsequently sequenced by whole-genome sequencing and subjected to pangenome analysis to retrieve data on the genotype, serotype, antimicrobial resistance (AMR), and virulence markers. Eight out of sixty-four Listeria spp. isolates were identified as L. monocytogenes. The serogroup analysis detected that 62.5% of the L. monocytogenes isolates belonged to serogroup IIa (1/2a and 3a) and 37.5% to serogroup IVb (4b, 4d, and 4e). Furthermore, the MLST (multilocus sequence typing) analysis of the eight detected L. monocytogenes isolates identified seven different sequence types (STs) and clonal complexes (CCs), i.e., ST1/CC1, ST2/CC2, ST6/CC6, ST7/CC7, ST21/CC21, ST504/CC475, and ST1413/CC739. The core genome MLST analysis also showed high allelic differences and suggests plant-specific isolates. Regarding the AMR, we detected phenotypic resistance against benzylpenicillin, fosfomycin, and moxifloxacin in all eight L. monocytogenes isolates. Moreover, virulence factors, such as prfA, hly, plcA, plcB, hpt, actA, inlA, inlB, and mpl, were identified in pathogenic and nonpathogenic Listeria species. The significance of L. monocytogenes in FNAO is growing and should receive increasing levels of attention.

Contrasting Genetic Diversity of Listeria Pathogenicity Islands 3 and 4 Harbored by Nonpathogenic Listeria spp.

Listeria monocytogenes causes the severe foodborne disease listeriosis. Several clonal groups of L. monocytogenes possess the pathogenicity islands Listeria pathogenicity island 3 (LIPI-3) and LIPI-4. Here, we investigated the prevalence and genetic diversity of LIPI-3 and LIPI-4 among 63 strains of seven nonpathogenic Listeria spp. from the natural environment, i.e., wildlife (black bears [Ursus americanus]) and surface water. Analysis of the whole-genome sequence data suggested that both islands were horizontally acquired but differed considerably in their incidence and genetic diversity. LIPI-3 was identified among half of the L. innocua strains in the same genomic location as in L. monocytogenes (guaA hot spot) in a truncated form, with only three strains harboring full-length LIPI-3, and a highly divergent partial LIPI-3 was observed in three Listeria seeligeri strains, outside the guaA hot spot. Premature stop codons (PMSCs) and frameshifts were frequently noted in the LIPI-3 gene encoding listeriolysin S. On the other hand, full-length LIPI-4 without any PMSCs was found in all Listeria innocua strains, in the same genomic location as L. monocytogenes and with ~85% similarity to the L. monocytogenes counterpart. Our study provides intriguing examples of genetic changes that pathogenicity islands may undergo in nonpathogenic bacterial species, potentially in response to environmental pressures that promote either maintenance or degeneration of the islands. Investigations of the roles that LIPI-3 and LIPI-4 play in nonpathogenic Listeria spp. are warranted to further understand the differential evolution of genetic elements in pathogenic versus nonpathogenic hosts of the same genus. IMPORTANCE Listeria monocytogenes is a serious foodborne pathogen that can harbor the pathogenicity islands Listeria pathogenicity island 3 (LIPI-3) and LIPI-4. Intriguingly, these have also been reported in nonpathogenic L. innocua from food and farm environments, though limited information is available for strains from the natural environment. Here, we analyzed whole-genome sequence data of nonpathogenic Listeria spp. from wildlife and surface water to further elucidate the genetic diversity and evolution of LIPI-3 and LIPI-4 in Listeria. While the full-length islands were found only in L. innocua, LIPI-3 was uncommon and exhibited frequent truncation and genetic diversification, while LIPI-4 was remarkable in being ubiquitous, albeit diversified from L. monocytogenes. These contrasting features demonstrate that pathogenicity islands in nonpathogenic hosts can evolve along different trajectories, leading to either degeneration or maintenance, and highlight the need to examine their physiological roles in nonpathogenic hosts.

Combination of growth conditions and InlB-specific dot-immunoassay for rapid detection of Listeria monocytogenes in raw milk

The gram-positive bacterium Listeria monocytogenes is an important foodborne pathogen contaminating dairy products. Closely related to L. monocytogenes saprophytic Listeria spp. are also frequent contaminators of food and, particularly, dairy products. To distinguish L. monocytogenes from nonpathogenic Listeria spp. and other bacteria, a dot-immunoassay was developed. The immunoassay is based on the polyclonal antibody to the secreted form of the surface virulence-associated L. monocytogenes-specific InlB protein. To increase InlB production, bacteria were grown on the brain-heart infusion agar supplemented with 0.2% activated charcoal (BHIC agar). Direct plating of artificially contaminated raw milk samples on the BHIC agar followed by the dot-immunoassay allowed a rapid identification of L. monocytogenes in concentrations as little as 10 cfu/mL. Using the developed approach, preliminary results were obtained within 14 h, and the final results were obtained after 26 h. The dot-immunoassay was tested on L. monocytogenes strains belonging to different clonal complexes and phylogenetic lineages, Listeria spp., and other bacterial species. Results showed the exceptional specificity of the developed dot-immunoassay for the rapid identification of L. monocytogenes.

Comparative Genomics Reveal the Utilization Ability of Variable Carbohydrates as Key Genetic Features of Listeria Pathogens in Their Pathogenic Lifestyles.

L. monocytogenes and L. ivanovii, the only two pathogens of Listeria, can survive in various environments, having different pathogenic characteristics. However, the genetic basis of their excellent adaptability and differences in pathogenicity has still not been completely elucidated. We performed a comparative genomic analysis based on 275 L. monocytogenes, 10 L. ivanovii, and 22 non-pathogenic Listeria strains. Core/pan-genome analysis revealed that 975 gene families were conserved in all the studied strains. Additionally, 204, 242, and 756 gene families existed uniquely in L. monocytogenes, L. ivanovii, and both, respectively. Functional annotation partially verified that these unique gene families were closely related to their adaptability and pathogenicity. Moreover, the protein-protein interaction (PPI) network analysis of these unique gene sets showed that plenty of carbohydrate transport systems and energy metabolism enzymes were clustered in the networks. Interestingly, ethanolamine-metabolic-process-related proteins were significantly enriched in the PPI network of the unique genes of the Listeria pathogens, which can be understood as a determining factor of their pathogenicity. The utilization capacity of multiple carbon sources of Listeria pathogens, especially ethanolamine, is the key genetic basis for their ability to adapt to various environments and pathogenic lifestyles.

Comparative Genomics of Listeria Species Recovered from Meat and Food Processing Facilities.

ABSTRACTListeria species (spp.) are contaminants that can survive in food, on equipment, and on food processing premises if appropriate hygiene measures are not used. Homologous stress tolerance genes, virulence gene clusters such as the prfA cluster, and clusters of internalin genes that contribute to the pathogenic potential of the strains can be carried by both pathogenic and nonpathogenic Listeria spp. To enhance understanding of the genome evolution of virulence and virulence-associated properties, a comparative genome approach was used to analyze 41 genome sequences belonging to L. innocua and L. welshimeri isolated from food and food processing facilities. Genetic determinants responsible for disinfectant and stress tolerance were identified, including the efflux cassette bcrABC and Tn6188_qac_1 disinfectant resistance determinant, and stress survival islets. These disinfectant-resistant genes were more frequently found in L. innocua (12%) than in L. welshimeri (2%). Several isolates representing the presumed nonpathogenic L. innocua still carried virulence-associated genes, including LGI2, LGI3, LIPI-3, and LIPI-4 which were absent in all L. welshimeri isolates. The mobile genetic elements identified were plasmids (pLGUG1 and J1776) and prophages (PHAGE_Lister_vB_LmoS_188, PHAGE_Lister_LP_030_3, PHAGE_Lister_A118, PHAGE_Lister_B054, and PHAGE_Lister_vB_LmoS_293). The results suggest that the presumed nonpathogenic isolates especially L. innocua can carry genes relevant to the strain’s virulence and stress tolerance in the food and food processing facilities.IMPORTANCE This study provides genomic insights into the recently expanded genus in order to gain valuable information about the evolution of the virulence and stress tolerance properties of the genus Listeria and the distribution of these genetic elements pertinent to the pathogenic potential across Listeria spp. and clonal lineages in South Africa (SA).

Abstract 4203: Tumor-targeted delivery of a neoantigen surrogate by non-pathogenic Listeria reduces metastatic ovarian cancer

Abstract Objectives: End-stage ovarian cancer is commonly characterized by intraperitoneal (IP) carcinomatosis and chemoresistance. To date, immune-mediated therapies have shown little success in this population in part due to the absence of neoantigens in these tumors and strong immune suppression in the tumor microenvironment (TME) preventing T cell activation. Our laboratory has developed a novel microbial-based immunotherapeutic strategy for selective delivery of a highly immunogenic tetanus toxoid protein (TT856-1313) into tumor cells through in vivo infection with attenuated Listeria monocytogenes. In mice previously vaccinated against TT, this leads to reactivation of pre-existing TT-specific memory T cells which kill the infected tumor cells in mouse models of pancreatic cancer. The addition of gemcitabine (GEM) improves T cell responses by reducing immune suppression in the TME. We hypothesize that the regimen Listeria-TT+GEM will have efficacy in vivo in mice with advanced ovarian cancer. Methods: Infection and killing of mouse and human ovarian tumor cells by Listeria-TT was analyzed in vitro. C57Bl/6 mice were vaccinated with the TT vaccine at six weeks of age. 3x106 luciferase-expressing ID8 syngeneic mouse tumor cells (Id8-Luc) were injected IP and tumor growth was monitored by Vivo Imaging Systems (IVIS.) Mice were divided into four treatment groups: saline, GEM, Listeria-TT, and Listeria-TT+GEM. Quantification of tumor burden was approximated by total flux (photons/second) within a region of interest (ROI) prior to, during, and at completion of therapy (days 0, 9, and 19 after tumor cell injection, respectively). Results: In vitro analyses demonstrated efficient infection and killing of mouse Id8-Luc and chemoresistant human Hey ovarian tumor cells by Listeria-TT. The response of tumors and metastases to the four prespecified treatment regimens was evaluated by IVIS as described above. On Day 0, there was no significant difference in the quantified tumor burden between the four treatment groups. On Days 9 and 19, the tumor burden was significantly less in the Listeria-TT+GEM group compared to the saline group (Mann-Whitney p=0.0159). Neither GEM nor Listeria-TT alone were significantly different from the saline group on day 9, but the effect of GEM was significantly different from saline on day 19 (p=0.0079). In a follow up study, the survival time of the Listeria-TT+GEM-treated mice was improved by 50% compared to the saline mice. The survival of GEM compared to saline is under investigation. Conclusions: Our results demonstrate that Listeria-TT+GEM strongly reduced metastatic ovarian cancer in mice. Additional experiments are ongoing to evaluate if more treatment cycles of Listeria-TT+GEM further improves the survival time. This study suggest that the combination therapy may be promising for human application. Citation Format: Lisa R. Gabor, Olivia R. Khouri, Ken Lin, Nicole Nevadunsky, Dennis Yi-Shin Kuo, Claudia Gravekamp. Tumor-targeted delivery of a neoantigen surrogate by non-pathogenic Listeria reduces metastatic ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4203.

Listeria innocua Biofilm Assay Using NanoLuc Luciferase.

Biofilms serve as a bacterial survival strategy, allowing bacteria to persist under adverse environmental conditions. The non-pathogenic Listeria innocua is used as a surrogate organism for the foodborne pathogen Listeria monocytogenes, because they share genetic and physiological similarities and can be used in a Biosafety Level 1 laboratory. Several methods are used to evaluate biofilms, including different approaches to determine biofilm biomass or culturability, viability, metabolic activity, or other microbial community properties. Routinely used methods for biofilm assay include the classical culture-based plate counting method, biomass staining methods (e.g., crystal violet and safranin red), DNA staining methods (e.g., Syto 9), methods that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts or resazurin), and PCR-based methods to quantify bacterial DNA. The NanoLuc (Nluc) luciferase biofilm assay is a viable alternative or complement to existing methods. Functional Nluc was expressed in L. innocua using the nisin-inducible expression system and bacterial detection was performed using furimazine as substrate. Concentration dependent bioluminescence signals were obtained over a concentration range greater than three log units. The Nluc bioluminescence method allows absolute quantification of bacterial cells, has high sensitivity, broad range, good day-to-day repeatability, and good precision with acceptable accuracy. The advantages of Nluc bioluminescence also include direct detection, absolute cell quantification, and rapid execution. Graphic abstract: Engineering Listeria innocua to express NanoLuc and its application in bioluminescence assay.

Accurate classification of Listeria species by MALDI-TOF mass spectrometry incorporating denoising autoencoder and machine learning

Listeria monocytogenes belongs to the category of facultative anaerobic bacteria, and is the pathogen of listeriosis, potentially lethal disease for humans. There are many similarities between L. monocytogenes and other non-pathogenic Listeria species, which causes great difficulties for their correct identification. The level of L. monocytogenes contamination in food remains high according to statistics from the Food and Drug Administration. This situation leads to food recall and destruction, which has caused huge economic losses to the food industry. Therefore, the identification of Listeria species is very important for clinical treatment and food safety. This work aims to explore an efficient classification algorithm which could easily and reliably distinguish Listeria species. We attempted to classify Listeria species by incorporating denoising autoencoder (DAE) and machine learning algorithms in matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, convolutional neural networks were used to map the high dimensional original mass spectrometry data to low dimensional core features. By analyzing MALDI-TOF MS data via incorporating DAE and support vector machine (SVM), the identification accuracy of Listeria species was 100%. The proposed classification algorithm is fast (range of seconds), easy to handle, and, more importantly, this method also allows for extending the identification scope of bacteria. The DAE model used in our research is an effective tool for the extraction of MALDI-TOF mass spectrometry features. Despite the fact that the MALDI-TOF MS dataset examined in our research had high dimensionality, the DAE + SVM algorithm was still able to exploit the hidden information embedded in the original MALDI-TOF mass spectra. The experimental results in our work demonstrated that MALDI-TOF mass spectrum combined with DAE + SVM could easily and reliably distinguish Listeria species.

Comparison of recombinant and synthetic listeriolysin- O peptide- based indirect ELISA vis-à-vis cultural isolation for detection of listeriosis in caprine and ovine species

The present study evaluated the comparative serodiagnostic efficacy of recombinant listeriolysin-O (rLLO) and synthetic LLO- 2 peptide-based indirect ELISA vis-à-vis cultural isolation using samples (n = 1326; blood, sera, vaginal swabs, and rectal swabs) collected from caprines (n = 350) and ovines (n = 50) having reproductive and/or nervous system disorders and/or healthy animals. On screening the test sera by rLLO- based ELISA, the antibodies against LLO (ALLO) were observed in 17.71% of the caprines and 2% of the ovines, respectively, while synthetic LLO-2- based ELISA revealed ALLO in 6.86% of caprines and not in ovines. Moreover, the adsorption of positive test sera with streptolysin-O (SLO) resulted in a significant reduction (7.43%; p < 0.05) in the seropositivity with rLLO- based ELISA, whereas LLO-2- based ELISA revealed marginal reduction (4.29%; p > 0.05) in the seropositivity. Overall, the seropositivity with LLO-2 synthetic peptide revealed comparatively less cross-reactivity in comparison to rLLO. The cultural isolation yielded five pathogenic L. monocytogenes isolates and three non-pathogenic Listeria spp. from caprine samples; however, Listeria spp. could not be recovered from any of the ovine samples. Further, on comparing seropositivity with the isolation study results, it was found that two out of the five animals from which pathogenic L. monocytogenes isolated were also found seropositive in both the ELISAs even after adsorption with SLO. Interestingly, rLLO- based ELISA detected antibodies against unadsorbed caprine sera even in those samples from which non-pathogenic Listeria spp. were isolated, whereas antibodies were not detected in LLO-2 peptide-based ELISA. In conclusion, it could be inferred that the synthetic LLO-2 peptide serves as a non- cross-reactive, ideal diagnostic antigen in serodiagnosis of capro-ovine listeriosis.

Comparison of Resampling Algorithms to Address Class Imbalance when Developing Machine Learning Models to Predict Foodborne Pathogen Presence in Agricultural Water

Recent studies have shown that predictive models can supplement or provide alternatives to E. coli-testing for assessing the potential presence of food safety hazards in water used for produce production. However, these studies used balanced training data and focused on enteric pathogens. As such, research is needed to determine 1) if predictive models can be used to assess Listeria contamination of agricultural water, and 2) how resampling (to deal with imbalanced data) affects performance of these models. To address these knowledge gaps, this study developed models that predict nonpathogenic Listeria spp. (excluding L. monocytogenes) and L. monocytogenes presence in agricultural water using various combinations of learner (e.g., random forest, regression), feature type, and resampling method (none, oversampling, SMOTE). Four feature types were used in model training: microbial, physicochemical, spatial, and weather. “Full models” were trained using all four feature types, while “nested models” used between one and three types. In total, 45 full (15 learners*3 resampling approaches) and 108 nested (5 learners*9 feature sets*3 resampling approaches) models were trained per outcome. Model performance was compared against baseline models where E. coli concentration was the sole predictor. Overall, the machine learning models outperformed the baseline E. coli models, with random forests outperforming models built using other learners (e.g., rule-based learners). Resampling produced more accurate models than not resampling, with SMOTE models outperforming, on average, oversampling models. Regardless of resampling method, spatial and physicochemical water quality features drove accurate predictions for the nonpathogenic Listeria spp. and L. monocytogenes models, respectively. Overall, these findings 1) illustrate the need for alternatives to existing E. coli-based monitoring programs for assessing agricultural water for the presence of potential food safety hazards, and 2) suggest that predictive models may be one such alternative. Moreover, these findings provide a conceptual framework for how such models can be developed in the future with the ultimate aim of developing models that can be integrated into on-farm risk management programs. For example, future studies should consider using random forest learners, SMOTE resampling, and spatial features to develop models to predict the presence of foodborne pathogens, such as L. monocytogenes, in agricultural water when the training data is imbalanced.

Listeria environmental sampling tests are compatible with polymorphic locus sequence typing.

Food processors invest significant resources into environmental sampling to detect contamination with potential pathogens, particularly Listeria monocytogenes. To facilitate these efforts, multiple environmental sampling tests (ESTs) have been developed and commercialized that minimize workload, turnaround time, and cost while providing convenient colorimetric detection. For presumptive-positive ESTs, we hypothesized that a relatively minor additional investment could provide, in addition to species confirmation, valuable strain typing data for tracking pathogen spread through a facility, identifying harborage sites, and distinguishing sporadic from persistent or resident contaminants. This hypothesis is based on the demonstrated compatibility of polymorphic locus sequence typing (PLST) with crude samples including food enrichments. Five Listeria ESTs were tested here: broth-based InSite (Hygiena), Path-Chek (Mericon), and Pathfinder (Hardy Diagnositics); and gel-based Petrifilm (3M) and HardyChrom (Hardy Diagnostics). ESTs were inoculated with strains representing two common L. monocytogenes serotypes and nonpathogenic Listeria innocua. Following incubation, broths or suspended colonies were heat treated to inactivate bacteria. Lysates or purified DNAs were prepared and used as templates in PCRs targeting the previously described PLST loci LmiMT1 and LisMT2. Single clear products were obtained from all inoculated ESTs; uninoculated controls were negative. PCR products were subjected to Sanger sequencing, yielding high-quality chromatograms. Phylogenetic analysis confirmed identities to previously determined sequences and revealed relatedness to serotype-matched strains represented in GenBank databases. PRACTICAL APPLICATION: Multiple environmental sampling tests have been commercialized in recent years to facilitate the proactive detection of pathogens, particularly Listeria monocytogenes, within food processing facilities. Coupling a positive detection test with strain typing would enhance its value by providing data that can be used to track pathogen spread through a facility, identify harborage sites, and distinguish sporadic from resident contamination.

Identification and Characterization of a Novel Genomic Island Harboring Cadmium and Arsenic Resistance Genes in Listeria welshimeri.

Listeria monocytogenes, the bacterial foodborne pathogen responsible for the severe disease listeriosis, frequently exhibits heavy metal resistance. Concurrent resistance to cadmium and arsenic in L. monocytogenes is strongly associated with the 35-kb chromosomal island LGI2. LGI2 has been encountered repeatedly among L. monocytogenes serotype 4b hypervirulent clones but, surprisingly, not among non-pathogenic Listeria spp. Here we describe a novel LGI2 variant, LGI2-3, in two L. welshimeri strains from an urban aquatic environment. Whole genome sequence analysis revealed that the genomes were closely related except for one prophage region and confirmed a chromosomally integrated LGI2-3. It harbored a cystathionine beta-lyase gene previously only encountered in LGI2-1 of L. monocytogenes clonal complex 1 but was otherwise most closely related to LGI2. LGI2-3 harbored a novel cadAC cassette (cadA7C7) that, like LGI2′s cadA4C4, was associated with lower-level tolerance to cadmium (MIC 50 μg/mL) than other cadAC cassettes (MIC ≥ 140 μg/mL). CadA sequence analysis identified two amino acids that may be important for mediating different levels of cadmium tolerance. Our findings clearly demonstrated the potential for LGI2-like islands to be harbored by non-pathogenic Listeria spp. and generate intriguing hypotheses on the genetic diversity mediated by this island and its transfer among Listeria spp.

Comparison of biofilm formation between non-pathogenic Listeria strains under different stress conditions

AbstractMicro-organisms can attach to food surfaces and develop biofilms which present a concern in food and environmental safety. The main goal of the current study was to investigate the biofilm formation of six non-pathogenic Listeria strains under different stress conditions using a microplate assay. The effect of the weak biofilm-forming non-pathogenic Listeria strains on the biofilm formation of a strong biofilm-forming pathogenic Listeria strain (Listeria monocytogenes #8) was also examined. Listeria innocua CCM4030, Listeria innocua 2885 and Listeria seeligeri/welshimeri 292 showed the same patterns of biofilm formation with increasing NaCl concentrations from 0.05 to 15%, but all the other strains showed a continuously decreasing trend of OD595 in the same conditions. This study showed that in the case of non-pathogenic Listeria strains, higher concentrations of NaCl do not present a stress condition that enhances biofilm formation. Decrease in pH inhibited biofilm formation for all the non-pathogenic Listeria strains. The weak biofilm forming non-pathogenic Listeria strains (Listeria innocua 2885 and Listeria innocua CCM4030) overgrew the strong biofilm-forming Listeria strain (Listeria monocytogenes #8) during biofilm formation. This phenomenon could be beneficial and potentially be used as a novel control strategy to prevent the colonization of the pathogenic Listeria at food processing facilities such as in meat industry.

Multiplex PCR for the Identification of Pathogenic Listeria in Flammulina velutipes Plant Based on Novel Specific Targets Revealed by Pan-Genome Analysis

Listeria spp. is an important foodborne disease agent, often found in the fresh mushroom (Flammulina velutipes) and its production environment. The aim of this study was to develop multiplex PCR for rapid identification of Listeria monocytogenes and Listeria ivanovii, and nonpathogenic Listeria in F. velutipes plants. Pan-genome analysis was first used to identify five novel Listeria-specific targets: one for the Listeria genus, one for L. monocytogenes, and three for L. ivanovii. Primers for the novel targets were highly specific in individual reactions. The detection limits were 103–104 CFU/mL, meeting the requirements of molecular detection. A mPCR assay for the identification of pathogenic Listeria, with primers targeting the novel genes specific for Listeria genus (LMOSLCC2755_0944), L. monocytogenes (LMOSLCC2755_0090), and L. ivanovii (queT_1) was then designed. The assay specificity was robustly verified by analyzing nonpathogenic Listeria and non-Listeria spp. strains. The determined detection limits were 2.0 × 103 CFU/mL for L. monocytogenes and 3.4 × 103 CFU/mL for L. ivanovii, for pure culture analysis. Further, the assay detected 7.6 × 104 to 7.6 × 100 CFU/10 g of pathogenic Listeria spiked into F. velutipes samples following 4–12 h enrichment. The assay feasibility was evaluated by comparing with a traditional culture-based method, by analyzing 129 samples collected from different F. velutipes plants. The prevalence of Listeria spp. and L. monocytogenes was 58.1% and 41.1%, respectively. The calculated κ factors for Listeria spp., L. monocytogenes, and L. ivanovii were 0.97, 0.97, and 1, respectively. The results of the novel mPCR assay were highly consistent with those of the culture-based method. The new assay thus will allow rapid, specific, and accurate detection and monitoring of pathogenic Listeria in food and its production environment.

Receptor-targeted engineered probiotics mitigate lethal Listeria infection

Probiotic bacteria reduce the intestinal colonization of pathogens. Yet, their use in preventing fatal infection caused by foodborne Listeria monocytogenes (Lm), is inconsistent. Here, we bioengineered Lactobacillusprobiotics (BLP) to express the Listeria adhesion protein (LAP) from a non-pathogenic Listeria (L. innocua) and a pathogenic Listeria (Lm) on the surface of Lactobacillus casei. The BLP strains colonize the intestine, reduce Lm mucosal colonization and systemic dissemination, and protect mice from lethal infection. The BLP competitively excludes Lm by occupying the surface presented LAP receptor, heat shock protein 60 and ameliorates the Lm-induced intestinal barrier dysfunction by blocking the nuclear factor-κB and myosin light chain kinase-mediated redistribution of the major epithelial junctional proteins. Additionally, the BLP increases intestinal immunomodulatory functions by recruiting FOXP3+T cells, CD11c+ dendritic cells and natural killer cells. Engineering a probiotic strain with an adhesion protein from a non-pathogenic bacterium provides a new paradigm to exclude pathogens and amplify their inherent health benefits.

Magnesium ion disrupts LAP surface re‐association of Listeria monocytogenes by dissociation of InlB

Listeria monocytogenes crosses intestinal epithelial barrier for the systemic spread, which is facilitated in part, by Listeria adhesion protein (LAP). LAP, a housekeeping enzyme called acetaldehyde alcohol dehydrogenase, is present in both pathogenic and nonpathogenic Listeria. LAP is secreted with the aid of SecA2 translocon, and its’ re‐association on the surface of pathogenic Listeria i.e., L. monocytogenes is critical for its function. However, the cell wall component(s) involved in LAP interaction is unknown, which was investigated.Using L. monocytogenes F4244 (WT), an isogenic lap− strain and purified recombinant LAP in the Caco‐2 cell adhesion assay and Western blot, we examined LAP re‐association on lap− cells. Extraneous added LAP restored adhesion capacity of lap−, but not of L. innocua to Caco‐2 cells, suggesting the presence of LAP‐interacting receptor(s) only on pathogenic bacteria.To locate LAP interaction partners, co‐immunoprecipitation of WT cell wall fraction using anti‐LAP mAb was analyzed by SDS‐PAGE. Selected protein bands were subjected to MALDI‐TOF protein identification showing involvement of internalin B (InlB).Next, we investigated the effect of MgCl2 pretreatment on LAP re‐association on the cell surface, cell‐free supernatant, and consequent bacterial adhesion. Mg2+ is a wall teichoic acid (WTA) charge neutralizing agent that reduced levels of InlB binding on the WT cell wall, resulting in surface disassociation of LAP. However, only partial surface reduction of LAP was observed in ΔinlB strain suggesting more than one ligand is involved.In conclusion, our results indicate that both InlB and WTA may participate in LAP re‐association on L. monocytogenes cell surface, which is essential for its pathogenic activities.Support or Funding InformationThe technical assistance of Uma Aryal, Valerie Ryan, and Aaron Gall and funding from USDA‐ARS project number 59‐8072‐6‐001 through the Center for Food Safety Engineering at Purdue University are acknowledged.LAP Surface Ligand Fishing ExpeditionAs the experiment scheme shown (A), several Co‐immunoprecipated candidates were visualized by Sliver staining. Red arrow point to a potential receptor around 70 kDa (B). Abundance of each identified protein was graphed according to relative protein abundance (C). Internalin B is the first protein present only in pathogenic strain.Figure 1MgCl2 Treatment Affects LAP Dissociation and Bacterial AdhesionWB protein analysis in Supernatant and Cell Wall Fraction (A), growth curve (B) and adhesion (C) of LmWT grow with additional MgCl2 in TSBYE.Figure 2