Aquatic environment drives the emergence of cell wall-deficient dormant forms in Listeria.

Stressed bacteria can enter a dormant viable but non-culturable (VBNC) state. VBNC pathogens pose an increased health risk as they are undetectable by growth-based techniques and can wake up back into a virulent state. Although widespread in bacteria, the mechanisms governing this phenotypic switch remain elusive. Here, we investigate the VBNC state transition in the human pathogen Listeria monocytogenes. We show that bacteria starved in mineral water become VBNC by converting into osmotically stable cell wall-deficient coccoid forms, a phenomenon that occurs in other Listeria species. We reveal the bacterial stress response regulator SigB and the autolysin NamA as major actors of VBNC state transition. We lastly show that VBNC Listeria revert to a walled and virulent state after passage in chicken embryos. Our study provides more detail on the VBNC state transition mechanisms, revealing wall-free bacteria naturally arising in aquatic environments as a potential survival strategy in hypoosmotic and oligotrophic conditions.

Developing an Agent-Based Model that Predicts Listeria spp. Transmission to Assess Listeria Control Strategies in Retail Stores.

Contamination of fresh produce with Listeria monocytogenes can occur throughout the supply chain, including at retail, where Listeria spp., including L. monocytogenes, may be introduced and spread via various routes. However, limited tools are available for retailers to assess practices that can enhance control of Listeria transmission to fresh produce. Therefore, we developed an agent-based model that can simulate Listeria transmission in retail produce sections to optimize environmental sampling programs and evaluate control strategies. A single retail store was used as a model environment, in which various routes of Listeria introduction into and transmission between environmental surfaces were modeled. Model prediction (i.e., Listeria prevalence) was validated using a published longitudinal study for all surfaces that were included in both the model and the validation data. Sensitivity analysis using the Partial Rank Correlation Coefficient showed that (i) initial Listeria concentration from incoming produce, (ii) transfer coefficient from produce to employee's hands, and (iii) transfer coefficient from consumer to produce were the top three parameters that were significantly (p < 0.0018) associated with the mean Listeria prevalence across all agents, suggesting that the accuracy of these parameters are important for prediction of overall Listeria prevalence at retail. Cluster analysis grouped agents with similar contamination patterns into six unique clusters; this information can be used to optimize the sampling plans for retail environments. Scenario analysis suggested that (i) more stringent supplier control as well as (ii) practices reducing Listeria transmission via consumer's hands may have the largest impact on reducing finished product contamination. Overall, we show that an agent-based model can serve as a foundational tool to help with decision-making on Listeria control strategies at retail.

mSphere of Influence: The power of observational research.

Katrina Velle is a cell biologist who uses microscopy to study amoebae. In this mSphere of Influence article, she reflects on how a classic paper on Listeria by Tilney and Portnoy made an impact on her by highlighting how much we can learn from simply looking at cells.

Application of Environmental Monitoring Programs and Root Cause Analysis to Identify and Implement Interventions to Reduce or Eliminate Listeria Populations in Apple Packinghouses.

Controlling Listeria in produce packinghouses can be challenging due to the large number of potential contamination routes. For example, repeated isolation of the same Listeria subtype in a packinghouse could indicate persistence in the packinghouse or reintroduction of the same Listeria from an upstream source. To improve understanding of Listeria transmission patterns in packinghouses, we performed a longitudinal study in four apple packinghouses, including testing of 1,339 environmental sponges and whole genome sequencing (WGS)-based characterization of 280 isolates. Root cause analysis and subsequent intervention implementation were also performed and assessed for effectiveness. Listeria prevalence among environmental sponges collected from the four packinghouses was 20% (range of 5-31% for individual packinghouses). Sites that showed high Listeria prevalence included drains, forklift tires and forks, forklift stops, and waxing area equipment frames. A total of 240/280 WGS-characterized isolates were represented in 41 clusters, each containing two or more isolates that differed by ≤50 high-quality single nucleotide polymorphisms (hqSNPs); 21 clusters were isolated from one packinghouse over ≥2 samplings (suggesting persistence or possibly reintroduction), while 11 clusters included isolates from >2 packinghouses, suggesting common upstream sources. Some interventions successfully (i) reduced Listeria detection on forklift tires and forks (across packinghouses) and (ii) mitigated packinghouse-specific Listeria issues (e.g., in catch pans). However, interventions that lacked enhanced equipment disassembly when persistence was suspected typically appeared to be unsuccessful. Overall, while our data suggest a combination of intensive environmental sampling with subtyping and root cause analysis can help identify effective interventions, implementation of effective interventions continues to be a challenge in packinghouses.

Stress resistance insights of 65 Listeria strains: Acidic, low temperature, and high salt environments.

Genetically, Listeria monocytogenes is closely related to non-L. monocytogenes (L. innocua, L. welshimeri, L. grayi, L. aquatica, and L. fleischimannii). This bacterium is well known for its resistance to harsh conditions including acidity, low temperatures, and high salt concentrations. This study explored the responses of 65 Listeria strains to stress conditions and characterized the prevalence of stress-related genes. The 65 Listeria strains were isolated from different environments and their viability was assessed in four different tests: independent tests for pH 3, 1 °C, and 5 % salt concentration and multiple resistance tests that combined pH 3, 1 °C, 5 % salt. From the data, the 65 strains were categorized into stress-resistant (56) or stress-sensitive groups (9), with approximately 4 log CFU/mL differences. The PCR assay analyzed the prevalence of two virulence genes prfA and inlA, and eight stress-related genes: three acid (gadB, gadC, and atpD), two low temperature (betL and opuCA) and three salt resistance genes (flaA, cysS, and fbp). Two low temperature (bet and opuCA) and salt resistance (fbp) genes were more prevalent in the stress-resistant strains than in the stress-sensitive Listeria group.

Antimicrobial resistance and prevalence of Listeria species from raw milk and dairy products in Burdur, Turkey.

OBJECTIVES: Worldwide, but especially in emerging nations, concerns about food safety pose a serious obstacle to societal and economic progress. This research aimed to examine the prevalence of Listeria spp. in raw milk and dairy products in Burdur, as well as the presence of genes associated with biofilm formation and antibiotic resistance in the isolates. METHODS: A total of 185 samples, including raw milk, curd, cream, butter, yogurt and cheese, were randomly collected in Burdur. The enrichment and isolation methods specified by the United States Department of Agriculture was used to identify Listeria species in milk and dairy product samples. Culture-positive strains were identified as Listeria genus and as species by PCR. Antibiotic susceptibility of the isolates was evaluated against 14 antibiotics using the disc diffusion technique (EUCAST). RESULTS: Of them, 2.2% (4/185) were positive for Listeria spp. Listeria species were isolated from cheese and yogurt samples. Two of them were Listeria innocua 1.1% (2/185), one was Listeria ivanovii 0.5% (1/185) and the other was Listeria welshimeri 0.5% (1/185). As a result of multiplex PCR of the biofilm genotypic marker luxS and flaA genes, the flaA gene was detected in three of four isolates, the luxS gene was detected in one isolate, and these two genes were not found in one isolate. Although all isolates were resistant to gentamicin and rifampicin, they also showed multidrug resistance. CONCLUSION: This study revealed that the diversity of prevalence of Listeria spp. in Burdur requires microbial risk assessment in the milk and dairy products value chain and the need to focus on the problem of multiple antibiotic resistance.

Antimicrobial Resistance Profiles of Listeria Species Recovered from Retail Outlets in Gauteng Province, South Africa.

The study determined the antimicrobial resistance (AMR) profiles of Listeria spp. (L. monocytogenes, L. innocua, and L. welshimeri) recovered from beef and beef products sold at retail outlets in Gauteng Province, South Africa. A total of 112 isolates of Listeria spp., including L. monocytogenes (37), L. innocua (65), and L. welshimeri (10), were recovered from beef and beef products collected from 48 retail outlets. Listeria spp. was recovered by direct selective plating following selective enrichment, and PCR was used to confirm and characterize recovered isolates. The disc diffusion method determined the resistance to 16 antimicrobial agents. All 112 isolates of Listeria spp. exhibited resistance to one or more antibiotics (P < 0.05). The prevalence of AMR in Listeria isolates was high for nalidixic acid (99.1%) and cefotaxime (80.4%) but low for gentamycin (2.7%), sulfamethoxazole-trimethoprim (3.6%), azithromycin (5.4%), and doxycycline (6.3%). Overall, for the three species of Listeria, the prevalence of resistance varied significantly only for streptomycin (P = 0.016) and tetracycline (P = 0.034). Multidrug-resistant isolates were detected in 75.7% (28/37), 61.5% (40/65), and 80% (8/10) isolates of L. monocytogenes, L. innocua, and L. welshimeri, respectively. The prevalence of AMR was significantly affected by the location and size of retail outlets, type of beef and beef products, and serogroups of L. monocytogenes. The high prevalence of AMR, particularly among the L. monocytogenes isolates, poses potential therapeutic implications for human consumers of contaminated beef products. There is, therefore, a need to regulate and enforce the use of antimicrobial agents in humans and animals in South Africa.

Genetic recombination-mediated evolutionary interactions between phages of potential industrial importance and prophages of their hosts within or across the domains of Escherichia, Listeria, Salmonella, Campylobacter, and Staphylococcus.

BACKGROUND: The in-depth understanding of the role of lateral genetic transfer (LGT) in phage-prophage interactions is essential to rationalizing phage applications for human and animal therapy, as well as for food and environmental safety. This in silico study aimed to detect LGT between phages of potential industrial importance and their hosts. METHODS: A large array of genetic recombination detection algorithms, implemented in SplitsTree and RDP4, was applied to detect LGT between various Escherichia, Listeria, Salmonella, Campylobacter, Staphylococcus, Pseudomonas, and Vibrio phages and their hosts. PHASTER and RAST were employed respectively to identify prophages across the host genome and to annotate LGT-affected genes with unknown functions. PhageAI was used to gain deeper insights into the life cycle history of recombined phages. RESULTS: The split decomposition inferences (bootstrap values: 91.3-100; fit: 91.433-100), coupled with the Phi (0.0-2.836E-12) and RDP4 (P being well below 0.05) statistics, provided strong evidence for LGT between certain Escherichia, Listeria, Salmonella, and Campylobacter virulent phages and prophages of their hosts. The LGT events entailed mainly the phage genes encoding for hypothetical proteins, while some of these genetic loci appeared to have been affected even by intergeneric recombination in specific E. coli and S. enterica virulent phages when interacting with their host prophages. Moreover, it is shown that certain L. monocytogenes virulent phages could serve at least as the donors of the gene loci, involved in encoding for the basal promoter specificity factor, for L. monocytogenes. In contrast, the large genetic clusters were determined to have been simultaneously exchanged by many S. aureus prophages and some Staphylococcus temperate phages proposed earlier as potential therapeutic candidates (in their native or modified state). The above genetic clusters were found to encompass multiple genes encoding for various proteins, such as e.g., phage tail proteins, the capsid and scaffold proteins, holins, and transcriptional terminator proteins. CONCLUSIONS: It is suggested that phage-prophage interactions, mediated by LGT (including intergeneric recombination), can have a far-reaching impact on the co-evolutionary trajectories of industrial phages and their hosts especially when excessively present across microbially rich environments.

A secondary model for the effect of pH on the variability in growth fitness of Listeria innocua strains.

Variability in microbial growth is a keystone of modern Quantitative Microbiological Risk Assessment (QMRA). However, there are still significant knowledge gaps on how to model variability, with the most common assumption being that variability is constant. This is implemented by an error term (with constant variance) added on top of the secondary growth model (for the square root of the growth rate). However, this may go against microbial ecology principles, where differences in growth fitness among bacterial strains would be more prominent in the vicinity of the growth limits than at optimal growth conditions. This study coins the term "secondary models for variability", evaluating whether they should be considered in QMRA instead of the constant strain variability hypothesis. For this, 21 strains of Listeria innocua were used as case study, estimating their growth rate by the two-fold dilution method at pH between 5 and 10. Estimates of between-strain variability and experimental uncertainty were obtained for each pH using mixed-effects models, showing the lowest variability at optimal growth conditions, increasing towards the growth limits. Nonetheless, the experimental uncertainty also increased towards the extremes, evidencing the need to analyze both sources of variance independently. A secondary model was thus proposed, relating strain variability and pH conditions. Although the modelling approach certainly has some limitations that would need further experimental validation, it is an important step towards improving the description of variability in QMRA, being the first model of this type in the field.

Effects of Fermentation Temperature, Drying Temperature, Caliber Size, Starter Culture, and Sodium Lactate on Listeria monocytogenes Inactivation During Salami Production.

The effect of fermentation and drying temperatures, caliber, and sodium lactate on Listeria monocytogenes inactivation was studied in salami, produced in a pilot scale, inoculated with 107 CFU/g of Listeria innocua ATCC® 33090 as a surrogate microorganism for L. monocytogenes. Fermentation temperature varied between 24 and 30°C, drying temperature between 14 and 20°C, caliber between 5.1 and 13.2 cm, and sodium lactate initial concentrations in salamis were 0 and 2%. L. innocua counts, pH and water activity were determined in salamis over time. Sodium lactate (2%) decreased pH drop and Listeria inactivation during fermentation. Baranyi & Roberts equation was used to fit the experimental data and to estimate, for each test condition, inactivation rate (k), initial (Y0), and final counts of L. innocua (YEND). Total inactivation was calculated as Y0 minus YEND (Y0-YEND). Then, using a Box Benkhen experimental design, a quadratic model for k and a two-factor interaction model (2FI) for Y0 - YEND were obtained as functions of fermentation temperature, drying temperature, and caliber size. The models predicted that maximum k and Y0 -YEND, -2.62 ± 0.14 log10 CFU/g/day and 4.5 ± 0.1 log10 CFU/g, respectively, would be obtained fermenting at 30°C and drying at 20°C regardless of caliber. Drying at 14°C allowed Listeria growth until a water activity (aw) of 0.92 was reached. Therefore, if initial Listeria contamination is high (3 log10 CFU/g), drying at low temperatures will compromise product safety.