Functional Genomics Identified Novel Genes Involved in Growth at Low Temperatures in Listeria monocytogenes.

Listeria monocytogenes (Lm) is a foodborne pathogen that can cause severe human illness. Standard control measures for restricting bacterial growth, such as refrigeration, are often inadequate as Lm grows well at low temperatures. To identify genes involved in growth at low temperatures, a powerful functional genomics method Tn-seq was performed in this study. This genome-wide screening comprehensively identified the known and novel genetic determinants involved in low-temperature growth. A novel gene lmo1366, encoding rRNA methyltransferase, was identified to play an essential role in Lm growth at 16°C. In contrast, the inactivation of lmo2301, a gene encoding the terminase of phage A118, significantly enhanced the growth of Lm at 16°C. The deletion of lmo1366 or lmo2301 resulted in cell morphology alterations and impaired the growth rate in milk and other conditions at low temperatures. Transcriptomic analysis revealed that the Δlmo1366 and Δlmo2301 mutants exhibited altered transcriptional patterns compared to the wild-type strain at 16°C with significant differences in genes involved in ribosome structural stability and function, and membrane biogenesis, respectively. This work uncovered novel genetic determinants involved in Lm growth at 16°C, which could lead to a better understanding of how bacteria survive and multiply at low temperatures. Furthermore, these findings could potentially contribute to developing novel antibacterial strategies under low-temperature conditions. IMPORTANCE Listeria monocytogenes is a Gram-positive pathogen that contributes to foodborne outbreaks due to its ability to survive at low temperatures. However, the genetic determinants of Lm involved in growth at low temperatures have not been fully defined. Here, the genetic determinants involved in the low-temperature growth of Lm were comprehensively identified on a genome-wide scale by Tn-seq. The gene lmo1366, encoding rRNA methyltransferase, was identified essential for growth under low-temperature conditions. On the other hand, the gene lmo2301, encoding terminase of phage A118, plays a negative role in bacterial growth at low temperatures. The transcriptomic analysis revealed the potential mechanisms. These findings lead to a better understanding of how bacteria survive and multiply at low temperatures and could provide unique targets for novel antibacterial strategies under low-temperature conditions.

RibU is an essential determinant of Listeria pathogenesis that mediates acquisition of FMN and FAD during intracellular growth.

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential riboflavin-derived cofactors involved in a myriad of redox reactions across all forms of life. Nevertheless, the basis of flavin acquisition strategies by riboflavin auxotrophic pathogens remains poorly defined. In this study, we examined how the facultative intracellular pathogen Listeria monocytogenes, a riboflavin auxotroph, acquires flavins during infection. A L. monocytogenes mutant lacking the putative riboflavin transporter (RibU) was completely avirulent in mice but had no detectable growth defect in nutrient-rich media. However, unlike wild type, the RibU mutant was unable to grow in defined media supplemented with FMN or FAD or to replicate in macrophages starved for riboflavin. Consistent with RibU functioning to scavenge FMN and FAD inside host cells, a mutant unable to convert riboflavin to FMN or FAD retained virulence and grew in cultured macrophages and in spleens and livers of infected mice. However, this FMN- and FAD-requiring strain was unable to grow in the gallbladder or intestines, where L. monocytogenes normally grows extracellularly, suggesting that these sites do not contain sufficient flavin cofactors to promote replication. Thus, by deleting genes required to synthesize FMN and FAD, we converted L. monocytogenes from a facultative to an obligate intracellular pathogen. Collectively, these data indicate that L. monocytogenes requires riboflavin to grow extracellularly in vivo but scavenges FMN and FAD to grow in host cells.

An oxidation resistant pediocin PA-1 derivative and penocin A display effective anti-Listeria activity in a model human gut environment.

Pediocin PA-1 is a class IIa bacteriocin that is particularly effective against the foodborne pathogen Listeria monocytogenes. The loss of activity of PA-1 pediocin due to methionine oxidation is one of the challenges that limit the wider application of the bacteriocin. In this study, we heterologously expressed an oxidation resistant form of pediocin PA-1, i.e., pediocin M31L, and compared its activity to that of native pediocin PA-1 and to penocin A, a pediocin-like bacteriocin that displays a narrower antimicrobial spectrum. Minimal inhibitory concentration assays revealed that pediocin M31L was as effective as PA-1 and more effective than synthetic penocin A against Listeria with negligible activity against a range of obligate anaerobic commensal gut bacterial species. The anti-Listeria activity of these pediocins was also assessed in a simulated human distal colon model assay using the L. monocytogenes, spiked at 6.5 ± 0.13 Log CFU/mL, as a bioindicator. At 24 h, pediocin M31L and penocin A (2.6 µM) reduced Listeria counts to 3.5 ± 0.4 and 3.64 ± 0.62 Log CFU/mL, respectively, whereas Listeria counts were considerably higher, i.e. 7.75 ± 0.43 Log CFU/mL, in the non-bacteriocin-containing control. Ultimately, it was established that synthetic penocin A and the stable pediocin M31L derivative, heterologously produced, display effective anti-Listeria activity in a human gut environment.

An antimicrobial peptide specifically active against Listeria monocytogenes is secreted by Bacillus pumilus SF214.

BACKGROUND: Members of the Bacillus genus produce a large variety of antimicrobial peptides including linear or cyclic lipopeptides and thiopeptides, that often have a broad spectrum of action against Gram-positive and Gram-negative bacteria. We have recently reported that SF214, a marine isolated strain of Bacillus pumilus, produces two different antimicrobials specifically active against either Staphylococcus aureus or Listeria monocytogenes. The anti-Staphylococcus molecule has been previously characterized as a pumilacidin, a nonribosomally synthesized lipopetide composed of a mixture of cyclic heptapeptides linked to fatty acids of variable length. RESULTS: Our analysis on the anti-Listeria molecule of B. pumilus SF214 indicated that it is a peptide slightly smaller than 10 kDa, produced during the exponential phase of growth, stable at a wide range of pH conditions and resistant to various chemical treatments. The peptide showed a lytic activity against growing but not resting cells of Listeria monocytogenes and appeared extremely specific being inactive also against L. innocua, a close relative of L. monocytogenes. CONCLUSIONS: These findings indicate that the B. pumilus peptide is unusual with respect to other antimicrobials both for its time of synthesis and secretion and for its strict specificity against L. monocytogenes. Such specificity, together with its stability, propose this new antimicrobial as a tool for potential biotechnological applications in the fight against the dangerous food-borne pathogen L. monocytogenes.

Growth behavior of low populations of Listeria monocytogenes on fresh-cut mango, melon and papaya under different storage temperatures.

The growth behavior of Listeria monocytogenes low population (1-4 cells/sample) on fresh-cut mango, melon, papaya and fruit mix stored at 4, 8, 12 and 16 °C was evaluated over 10 days. Mango showed the lowest counts for L. monocytogenes during 10 days regardless of storage temperature (<1.7 log cfu.g-1). Melon supported high bacterial growth over 10 days, reaching 5 log cfu.g-1 at 16 °C. Both the fruit and storage temperature influenced the Listeria low population growth potential (δ). Cumulative frequency distribution of L. monocytogenes showed that after 10 days, 100% of fresh-cut fruits and fruit mix stored at 4 °C remained ≤2 log cfu.g-1, while at 12 and 16 °C 100% of melon, papaya and fruit mix samples exceeded this limit. At 8 °C, 100% of mango and fruit mix samples remained below this limit after 10 days, whereas 100% of melon and papaya reached it after 7 days. Results indicate 4 °C as the ideal to store safely fresh-cut mango, melon, papaya and fruit mix for 10 days. Besides, 8 °C can also be an option, but not for melon and papaya. Findings highlight the ability of L. monocytogenes to survive and grow in fresh-cut fruits even at a very low initial population levels.

Listeria monocytogenes Sublethal Injury and Viable-but-Nonculturable State Induced by Acidic Conditions and Disinfectants.

The dormancy continuum hypothesis states that in response to stress, cells enter different stages of dormancy ranging from unstressed living cells to cell death, in order to ensure their long-term survival under adverse conditions. Exposure of Listeria monocytogenes cells to sublethal stressors related to food processing may induce sublethal injury and the viable-but-nonculturable (VBNC) state. In this study, exposure to acetic acid (AA), hydrochloric acid (HCl), and two disinfectants, peracetic acid (PAA) and sodium hypochlorite (SH), at 20°C and 4°C was used to evaluate the potential induction of L. monocytogenes strain Scott A into different stages of dormancy. To differentiate the noninjured subpopulation from the total population, tryptic soy agar with 0.6% yeast extract (TSAYE), supplemented or not with 5% NaCl, was used. Sublethally injured and VBNC cells were detected by comparing plate counts obtained with fluorescence microscopy and by using combinations of carboxyfluorescein and propidium iodide (viable/dead cells). Induction of sublethal injury was more intense after PAA treatment. Two subpopulations were detected, with phenotypes of untreated cells and small colony variants (SCVs). SCVs appeared as smaller colonies of various sizes and were first observed after 5 min of exposure to 5 ppm PAA at 20°C. Increasing the stress intensity from 5 to 40 ppm PAA led to earlier detection of SCVs. L. monocytogenes remained culturable after exposure to 20 and 30 ppm PAA for 3 h. At 40 ppm, after 3 h of exposure, the whole population was considered nonculturable, while cells remained metabolically active. These results corroborate the induction of the VBNC state. IMPORTANCE Sublethally injured and VBNC cells may evade detection, resulting in underestimation of a food product's microbial load. Under favorable conditions, cells may regain their growth capacity and acquire new resistant characteristics, posing a major threat for public health. Induction of the VBNC state is crucial for foodborne pathogens, such as L. monocytogenes, the detection of which relies almost exclusively on the use of culture recovery techniques. In the present study, we confirmed that sublethal injury is an initial stage of dormancy in L. monocytogenes that is followed by the VBNC state. Our results showed that PAA induced SCVs (a phenomenon potentially triggered by external factors) and the VBNC state in L. monocytogenes, indicating that tests of lethality based only on culturability may provide false-positive results regarding the effectiveness of an inactivation treatment.

Antibacterial Properties of Melanoidins Produced from Various Combinations of Maillard Reaction against Pathogenic Bacteria.

Novel melanoidins are produced by the Maillard reaction. Here, melanoidins with high antibacterial activity were tested by examining various combinations of reducing sugars and amino acids as reaction substrates. Twenty-two types of melanoidins were examined by combining two reducing sugars (glucose and xylose) and eleven l-isomers of amino acids (alanine, arginine, glutamine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine) to confirm the effects of these melanoidins on the growth of Listeria monocytogenes at 25°C. The melanoidins produced from the combination of d-xylose with either l-phenylalanine (Xyl-Phe) or l-proline (Xyl-Pro), for which absorbance at 420 nm was 3.5 ± 0.2, completely inhibited the growth of L. monocytogenes at 25°C for 48 h. Both of the melanoidins exhibited growth inhibition of L. monocytogenes which was equivalent to the effect of nisin (350 IU/mL). The antimicrobial spectrum of both melanoidins was also investigated for 10 different species of bacteria, including both Gram-positive and Gram-negative species. While Xyl-Phe-based melanoidin successfully inhibited the growth of Bacillus cereus and Brevibacillus brevis, Xyl-Pro-based melanoidin inhibited the growth of Salmonella enterica Typhimurium. However, no clear trend in the antimicrobial spectrum of the melanoidins against different bacterial species was observed. The findings in the present study suggest that melanoidins generated from xylose with phenylalanine and/or proline could be used as potential novel alternative food preservatives derived from food ingredients to control pathogenic bacteria. IMPORTANCE Although the antimicrobial effect of melanoidins has been reported in some foods, there have been few comprehensive investigations on the antimicrobial activity of combinations of reaction substrates of the Maillard reaction. The present study comprehensively investigated the potential of various combinations of reducing sugars and amino acids. Because the melanoidins examined in this study were produced simply by heating in an autoclave at 121°C for 60 min, the targeted melanoidins can be easily produced. The melanoidins produced from combinations of xylose with either phenylalanine or proline exhibited a wide spectrum of antibiotic effects against various pathogens, including Listeria monocytogenes, Bacillus cereus, and Salmonella enterica Typhimurium. Since the antibacterial effect of the melanoidins on L. monocytogenes was equivalent to that of a nisin solution (350 IU/mL), we might expect a practical application of melanoidins as novel food preservatives.

Non-coding RNA regulates phage sensitivity in Listeria monocytogenes.

Non-coding RNAs (ncRNAs) have gained increasing attention as their diverse roles in virulence and environmental stress in Listeria monocytogenes have become clearer. The ncRNA rliB is an atypical member of the CRISPR family, conserved at the same genomic locus in all analyzed L. monocytogenes genomes and also in other Listeria species. In this study, rliB defective mutants (Lm3-22-ΔrliB) were constructed by homologous recombination. The growth cycle of Lm3-22-ΔrliB mutants was slower than that of wild-type Lm3-22. The sensitivity of Lm3-22-ΔrliB to the Listeria phage vB-LmoM-SH3-3 was significantly increased, and the efficiency of plaque formation was enhanced by 128 fold. Compared with wild type, the adhesion and invasion of Lm3-22-ΔrliB decreased significantly (9.3% and 1.33%, respectively). After 4 hours of infection, the proliferation of Lm3-22-ΔrliB in RAW264.7 cells also decreased significantly. Transcription level of invasion-related surface proteins showed that the internalin genes lmo0610 and lm0514, and the peptidoglycan binding protein gene lmo1799 in Lm3-22-ΔrliB were significantly increased. In addition, after interaction with phage, the transcription levels of inlA, lmo0610, lmo1799, lmo2085, and lmo0514 in Lm3-22-ΔrliB cells were significantly upregulated, while inlB was downregulated, compared with Lm3-22 control group with phage treatment. Therefore, rliB deletion effectively regulated the interaction between Listeria and phage, weaken its invasion ability, and provided a new theoretical basis for biocontrol of phage.

Plants as a realized niche for Listeria monocytogenes.

Listeria monocytogenes is a human pathogen. It is the causative agent of listeriosis, the leading cause of bacterial-linked foodborne mortality in Europe and elsewhere. Outbreaks of listeriosis have been associated with the consumption of fresh produce including vegetables and fruits. In this review we summarize current data providing direct or indirect evidence that plants can serve as habitat for L. monocytogenes, enabling this human pathogen to survive and grow. The current knowledge of the mechanisms involved in the interaction of this bacterium with plants is addressed, and whether this foodborne pathogen elicits an immune response in plants is discussed.

Heat Inactivation of Listeria monocytogenes on Pecans, Macadamia Nuts, and Sunflower Seeds.

This project was undertaken to determine the kinetic parameters of thermal inactivation of Listeria monocytogenes on pecans, macadamia nuts, and sunflower seeds subjected to heat treatments simulating industry processes. Five strains were grown in nonselective medium, mixed, and resuspended before inoculating macadamia nuts, pecans, and sunflower seeds (6 to 9 Log CFU/g). Redried inoculated pecans and macadamia nuts were heated in an oven at a temperature range of 90 to 140°C. Unshelled sunflower seeds were heated in sunflower seed oil. The thermal inactivation was determined by measuring viable cell counts using standard microbiological methods. Average count data were fit to the log-linear model, and thermal-death kinetics were calculated. On pecans, the viable Listeria counts were reduced by 3 and 3.5 Log CFU/g after 40 min at 110°C and 8 min at 140°C, respectively. On macadamia nuts, the L. monocytogenes population was reduced by 5 Log CFU/g after 20 min at 120°C. Unshelled sunflower seeds were subjected to heat treatment via a hot-oil bath. On sunflower seeds, >7 Log CFU/g reductions were observed after 15 min at 120°C. The thermal resistance (D value) for inactivation on pecans at 140°C was 3.1 min and on macadamia nuts at 120°C was 4.4 min. The inactivation of L. monocytogenes was influenced by the kind of nut or seed. These results suggest that L. monocytogenes has a relatively high thermal tolerance. The findings from this study will contribute to the assessment of the effectiveness of heat treatment for control of this pathogen on nuts and seeds. IMPORTANCE Listeria monocytogenes is a major concern for the food industry in ready-to-eat (RTE) foods. In recent years, large-scale recalls have occurred with contaminated sunflower seeds and macadamia nuts that triggered product withdrawals. These events stress the importance of understanding Listeria's ability to survive heat treatments in these low-water activity foods. Nuts and seeds are subjected to a variety of thermal treatments typically referred as roasting. To date, no listeriosis outbreak has been linked to nuts and seeds, but the recent recognition that this pathogen can be detected in commercial products stresses the need for research on thermal treatments. The characterization of heat inactivation kinetics at temperatures typically used during roasting processes will be very beneficial for validation studies. This manuscript reports inactivation rates of L. monocytogenes strains inoculated onto macadamia nuts, sunflower seeds, and pecan halves subjected to temperatures between 90 and 140°C.

Isolation and Identification of Dominant Bacteria from Raw Donkey Milk Produced in a Region of Morocco by QIIME 2 and Evaluation of Their Antibacterial Activity.

Recently, the interest in donkey milk has increased considerably because it proved high nutritive and functional values of their ingredients. Its chemical composition is widely studied, but its microbiota, especially lactic acid bacteria, remains less studied. This study focuses on analyzing, isolating, and identifying lactic acid bacteria and evaluating their capacity to produce biomolecules with antibacterial activity. Among 44 strains identified, 43 are Gram-positive, and most are catalase-negative and cocci-shaped. Five strains were selected to evaluate their antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli. Different induction methods allowed to amplify the antibacterial effects against these pathogenic strains.

The Digestibility of Hibiscus sabdariffa L. Polyphenols Using an In Vitro Human Digestion Model and Evaluation of Their Antimicrobial Activity.

Hibiscus sabdariffa L. (H.s.) is a polyphenolic-rich plant commonly consumed either as a beverage or spice. The aim of the present study was to evaluate the in vitro digestibility of H.s. polyphenols using an in vitro model of digestion which simulates the human stomach and small intestine. The bioaccessible polyphenols released in the digested samples were analyzed by liquid chromatography coupled to photodiode array and mass spectrometry detection. H.s. anthocyanins (cyanidin-3-O-sambubioside and delphinidin-3-O-sambubioside) content drastically dropped during the digestion process from 2.91 ± 0.03 µg g-1 and 8.53 ± 0.08 µg g-1 (w/w) CG (Cyanidin-glucoside) in the raw extract, respectively, to 0.12 ± 0.01 µg g-1 0.12 ± 0.01 µg g-1 (w/w) CG at the end of duodenal digestion. Total polyphenols also have shown a decrease from 1192.65 ± 30.37 µg g-1 (w/w) in the raw extract to 282.24 ± 7.21 µg g-1 (w/w) by the end of gastric digestion, in contrast to their increase by the end of duodenal digestion 372.91 ± 3.97 µg g-1 (w/w). On the other hand, the decrease in certain compounds (e.g., caffeoylquinicandcoumaroylquinic acids) was observed during gastric digestion resulting in an increase of quinic acid in the duodenal aliquots, thus suggesting that this compound was derived from the degradation of the more complex hydroxycinnamic acids. H.s. extract also exhibited a bacteriostatic effect against Staphylococcus aureus ATCC 6538 (MIC of 2.5 mg mL-1) and a bactericidal effect against a food isolate of Listeria monocytogenes (MBC of 2.5 mg mL-1). The undigested polyphenols of H.s. in the upper gastrointestinal tract enters the colon, where they are metabolized by the gut microbiota. The present study results showed that resistance of H.s. polyphenols during gastrointestinal digestion might affect their uptake, resulting in a decrease in their digestibility.

Low-energy X-ray inactivation of Listeria monocytogenes in mono-/co-culture biofilms with Pseudomonas fluorescens on food contact surfaces.

This study assessed the inactivation kinetics of 150 keV low-energy X-ray on mono-/co-culture biofilms of Listeria monocytogenes and Pseudomonas fluorescens on three different food-contact-surfaces (polyethylene, acrylic, and stainless steel). The results indicated that the level of biofilm formation of mono-/co-cultures of L. monocytogenes and P. fluorescens was the highest on acrylic. The mono-culture L. monocytogenes biofilm cells exhibited higher resistance to the low-energy X-rays than the corresponding mono-culture P. fluorescens biofilm cells, regardless of surface types. Furthermore, co-culture had enhanced the resistance of both P. fluorescens and L. monocytogenes biofilm cells to the low-energy X-ray. Two kinetic models for the inactivation process were investigated, including (i) Linear model and (ii) Weibull model. Based on R2, RMSE and AIC analysis, the Weibull model was superior in fitting the inactivation curves of low-energy X-ray on L. monocytogenes in mono-/co-culture biofilms with P. fluorescens. For mono-culture biofilms, the irradiation achieved the tR1 value (derived from the Weibull model, i.e., the dose required for the first 1-log reduction) of 46.36-50.81 Gy for L. monocytogenes and the tR1 value of 25.61-31.33 Gy for P. fluorescens. For co-culture biofilms, higher tR1 values for L. monocytogenes (59.54-70.77 Gy) and P. fluorescens (32.73-45.13 Gy) were yielded than those for their individual counterparts in mono-culture biofilm.

Study of the microbial diversity of a panel of Belgian artisanal cheeses associated with challenge studies for Listeria monocytogenes.

High throughput sequencing could become a powerful tool in food safety. This study was the first to investigate artisanal cheeses from Belgium (31 batches) using metagenetics, in relation to Listeria monocytogenes growth data acquired during a previous project. Five cheese types were considered, namely unripened acid-curd cheeses, smear- and mold-ripened soft cheeses, and Gouda-type and Saint-Paulin-type cheeses. Each batch was analyzed in triplicate the first and the last days of storage at 8 °C. Globally, 2697 OTUs belonging to 277 genera and to 15 phyla were identified. Lactococcus was dominant in all types, but Streptococcus was co-dominant in smear-ripened soft cheeses and Saint-Paulin-type cheeses. The dominant population was not always associated with added starter cultures. Bacterial richness and diversity were significantly higher in both types of soft cheeses than in other categories, including particular genera like Prevotella, Faecalibacterium and Hafnia-Obesumbacterium in mold-ripened cheeses and Brevibacterium, Brachybacterium, Microbacterium, Bacteroides, Corynebacterium, Marinilactibacillus, Fusobacterium, Halomonas and Psychrobacter in smear-ripened soft cheeses. A strong correlation was observed between no growth of L. monocytogenes in a smear-ripened cheese and the presence of an unknown Fusobacterium (relative abundance around 10%). This in silico correlation should be confirmed by further experiments in vitro and in situ.

Peroxyacetic acid and chlorine dioxide unlike chlorine induce viable but non-culturable (VBNC) stage of Listeria monocytogenes and Escherichia coli O157:H7 in wash water.

The elaboration of guidelines for the industry to establish minimum concentration to prevent cross-contamination during washing practices based on operational limits is the core of the recommended criteria for the use of sanitizers. Several studies have evidenced that sanitizers reduced the levels of foodborne pathogens. However, they might lead to the progress into a viable but non-culturable (VBNC) state of the cells. This evidence has raised concerns regarding the effectiveness of the recommended washing practices for the inactivation of microbial cells present in the process wash water (PWW). The present study evaluated if the most commonly used sanitizers, including sodium hypochlorite (chlorine), peroxyacetic acid (PAA) and chlorine dioxide (ClO2) at established operational limits induced the VBNC stage of Listeria monocytogenes and Escherichia coli O157:H7. Prevention of cross-contamination was examined in four different types of PWW from washing shredded lettuce and cabbage, diced onions, and baby spinach under simulated commercial conditions of high organic matter and 1 min contact time. The results obtained for chlorine showed that recommended operational limits (20-25 mg/L free chlorine) were effective in inactivating L. monocytogenes and E. coli O157:H7 in the different PWWs. However, the operational limits established for PAA (80 mg/L) and ClO2 (3 mg/L) reduced the levels of culturable pathogenic bacteria but induced the VBNC state of the remaining cells. Consequently, the operational limits for chlorine are satisfactory to inactivate foodborne pathogens present in PWW and prevent cross-contamination but higher concentrations or longer contact times should be needed for PAA and ClO2 to reduce the likelihood of the induction of VBNC bacteria cells, as it represents a hazard.

High throughput screening of technological and biopreservation traits of a large set of wild lactic acid bacteria from Brazilian artisanal cheeses.

This study aimed to evaluate technological (acidification, proteolysis, lipolysis, resistance to low pH, NaCl, and bile salts) and biopreservation (antimicrobial activity against foodborne pathogens) features of 1002 LAB by high throughput screening (HTS) methods. The LAB was isolated from 11 types of Brazilian artisanal cheeses (BAC) marketed in the main 5 producing regions. Remarkable intra-species variability in acidification rates have been found, which was most pronounced between isolates from Mina's artisanal cheeses, Caipira and Coalho cheeses. Lacticaseibacillus paracasei and Levilactobacillus brevis showed the fastest acidification rate; however, all isolates showed slower acidification rates than a lactococcal control strain (4.3 × lower). When testing inhibitory effects, > 75% of LAB isolates could inhibit the growth of Staphylococcus aureus ATCC 19095 and Listeria monocytogenes ATCC 7644. Two of these isolates, identified as Lactiplantibacillus plantarum and Lentilactobacillus buchneri, the sterile and neutral supernatants alone, were sufficient to inhibit L. monocytogenes growth. Principal component analysis (PCA) allowed the identification of functional groups based on proteolytic and lipolytic activity, osmotic stress resistance, and inhibition of L. monocytogenes. The type of cheese the isolates were recovered from influenced properties such as anti-listerial compounds and lipolytic enzyme production. The use of HTS and multivariate statistics allowed insights into a diverse set of LAB technological and biopreservation properties. These findings allow a profound knowledge of the heterogeneity of a large set of isolates, which can be further used to design starter cultures with varied and combined properties, such as biopreservation and technological features. Besides that, HTS makes it possible to analyze a vast panel of LAB strains, reducing costs and time within laboratory analysis, while avoiding the loss of information once all LAB are tested at the same time (differently from the traditional labor-intensive approach, in which a few numbers of strains is tested per time).

Anaerobic Growth of Listeria monocytogenes on Rhamnose Is Stimulated by Vitamin B12 and Bacterial Microcompartment-Dependent 1,2-Propanediol Utilization.

The foodborne pathogen Listeria monocytogenes can form proteinaceous organelles called bacterial microcompartments (BMCs) that optimize the utilization of substrates, such as 1,2-propanediol, and confer an anaerobic growth advantage. Rhamnose is a deoxyhexose sugar abundant in a range of environments, including the human intestine, and can be degraded in anaerobic conditions into 1,2-propanediol, next to acetate and lactate. Rhamnose-derived 1,2-propanediol was found to link with BMCs in some human pathogens such as Salmonella enterica, but the involvement of BMCs in rhamnose metabolism and potential physiological effects on L. monocytogenes are still unknown. In this study, we first test the effect of rhamnose uptake and utilization on anaerobic growth of L. monocytogenes EGDe without or with added vitamin B12, followed by metabolic analysis. We show that vitamin B12-dependent activation of pdu stimulates metabolism and anaerobic growth of L. monocytogenes EGDe on rhamnose via 1,2-propanediol degradation into 1-propanol and propionate. Transmission electron microscopy of pdu-induced cells shows that BMCs are formed, and additional proteomics experiments confirm expression of pdu BMC shell proteins and enzymes. Finally, we discuss the physiological effects and energy efficiency of L. monocytogenes pdu BMC-driven anaerobic rhamnose metabolism and the impact on competitive fitness in environments such as the human intestine. IMPORTANCE Listeria monocytogenes is a foodborne pathogen causing severe illness and, as such, it is crucial to understand the molecular mechanisms contributing to its survival strategy and pathogenicity. Rhamnose is a deoxyhexose sugar abundant in a range of environments, including the human intestine, and can be degraded in anaerobic conditions into 1,2-propanediol. In our previous study, the utilization of 1,2-propanediol (pdu) in L. monocytogenes was proved to be metabolized in bacterial microcompartments (BMCs), which are self-assembling subcellular proteinaceous structures and analogs of eukaryotic organelles. Here, we show that the vitamin B12-dependent activation of pdu stimulates metabolism and anaerobic growth of L. monocytogenes EGDe on rhamnose via BMC-dependent 1,2-propanediol utilization. Combined with metabolic and proteomics analysis, our discussion on the physiological effects and energy efficiency of BMC-driven rhamnose metabolism shed new light to understand the impact on L. monocytogenes competitive fitness in ecosystems such as the human intestine.

Investigating the Roles of Listeria monocytogenes Peroxidases in Growth and Virulence.

Bacteria have necessarily evolved a protective arsenal of proteins to contend with peroxides and other reactive oxygen species generated in aerobic environments. Listeria monocytogenes encounters an onslaught of peroxide both in the environment and during infection of the mammalian host, where it is the causative agent of the foodborne illness listeriosis. Despite the importance of peroxide for the immune response to bacterial infection, the strategy by which L. monocytogenes protects against peroxide toxicity has yet to be illuminated. Here, we investigated the expression and essentiality of all the peroxidase-encoding genes during L. monocytogenes growth in vitro and during infection of murine cells in tissue culture. We found that chdC and kat were required for aerobic growth in vitro, and fri and ahpA were each required for L. monocytogenes to survive acute peroxide stress. Despite increased expression of fri, ahpA, and kat during infection of macrophages, only fri proved necessary for cytosolic growth. In contrast, the proteins encoded by lmo0367, lmo0983, tpx, lmo1609, and ohrA were dispensable for aerobic growth, acute peroxide detoxification, and infection. Together, our results provide insight into the multifaceted L. monocytogenes peroxide detoxification strategy and demonstrate that L. monocytogenes encodes a functionally diverse set of peroxidase enzymes. IMPORTANCE Listeria monocytogenes is a facultative intracellular pathogen and the causative agent of the foodborne illness listeriosis. L. monocytogenes must contend with reactive oxygen species generated extracellularly during aerobic growth and intracellularly by the host immune system. However, the mechanisms by which L. monocytogenes defends against peroxide toxicity have not yet been defined. Here, we investigated the roles of each of the peroxidase-encoding genes in L. monocytogenes growth, peroxide stress response, and virulence in mammalian cells.

Characterization of Pediococcus acidilactici postbiotic and impact of postbiotic-fortified chitosan coating on the microbial and chemical quality of chicken breast fillets.

This study was carried out to characterize antioxidant activity, total phenolic content, and the phenolic and flavonoids profile of postbiotic of Pediococcus acidilactici and to evaluate the effects of postbiotics (10% and 50%) alone and in combination with chitosan coating (1%) on the microbial and chemical quality of chicken breast fillets during storage at 4 °C. Antioxidant activity and total phenolic content of the postbiotics were found to be 1291.02 ± 1.5 mg/L TEAC and 2336.11 ± 2.36 mg/L GAE, respectively. The most abundant phenolic was vanillic acid, followed by t-caffeic, gallic, and caftaric acids. The postbiotic-chitosan (50% + 1%) combination decreased L. monocytogenes and S. Typhimurium counts by 1.5 and 2.1 log10 CFU/g, respectively, compared to the control (P < 0.05). This combination decreased the total viable count (TVC), lactic acid bacteria (LAB), and psychrotrophic bacteria count compared to the control (P < 0.05). No differences were found in thiobarbituric acid (TBA) values among the samples during storage (P > 0.05). Postbiotic treatment did not significantly change the pH values and color properties of the breast fillets (P > 0.05). Postbiotic-chitosan combinations extended the shelf-life by up to 12 days compared to the control. In conclusion, the postbiotic-chitosan combination can be used to preserve and improve the microbial quality of chicken meat products.

Function and distribution of the conjugative plasmid pLM1686 in foodborne Listeria monocytogenes in China.

Listeria monocytogenes, a fatal foodborne pathogen has the extraordinary capacity to survive in harsh conditions and is a potential threat to public health. A novel 91 kb plasmid pLM1686 was found in the prevalent L. monocytogenes sequence type (ST) 87 strain in China. In this study, the function and distribution of pLM1686 were firstly investigated in L. monocytogenes. The results showed plasmid pLM1686 had self-transmissible ability and existed in various types of L. monocytogenes isolates belonging to two lineages (lineage I and II), four serotypes (1/2b, 3b, 1/2c and 1/2a) and four STs (ST87, ST59, ST9 and ST120). The wild strain LM1686 and transconjugant strain 10403SP1686 exhibited significantly higher growth rate and biofilm formation in Modification of Welshimer's medium (MWB), greater salinity tolerance, stronger cell invasion and higher cytotoxicity than plasmid-cured strain and reference strain 10403S. Moreover, plasmid curing caused the loss of cadmium resistance of strain, and the recipient strain acquired cadmium resistance after conjugation. Thus, pLM1686 would provide L. monocytogenes advantages of surviving in adverse environments.