Cas13-induced cellular dormancy prevents the rise of CRISPR-resistant bacteriophage.

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci in prokaryotes are composed of 30-40-base-pair repeats separated by equally short sequences of plasmid and bacteriophage origin known as spacers1-3. These loci are transcribed and processed into short CRISPR RNAs (crRNAs) that are used as guides by CRISPR-associated (Cas) nucleases to recognize and destroy complementary sequences (known as protospacers) in foreign nucleic acids4,5. In contrast to most Cas nucleases, which destroy invader DNA4-7, the type VI effector nuclease Cas13 uses RNA guides to locate complementary transcripts and catalyse both sequence-specific cis- and non-specific trans-RNA cleavage8. Although it has been hypothesized that Cas13 naturally defends against RNA phages8, type VI spacer sequences have exclusively been found to match the genomes of double-stranded DNA phages9,10, suggesting that Cas13 can provide immunity against these invaders. However, whether and how Cas13 uses its cis- and/or trans-RNA cleavage activities to defend against double-stranded DNA phages is not understood. Here we show that trans-cleavage of transcripts halts the growth of the host cell and is sufficient to abort the infectious cycle. This depletes the phage population and provides herd immunity to uninfected bacteria. Phages that harbour target mutations, which easily evade DNA-targeting CRISPR systems11-13, are also neutralized when Cas13 is activated by wild-type phages. Thus, by acting on the host rather than directly targeting the virus, type VI CRISPR systems not only provide robust defence against DNA phages but also prevent outbreaks of CRISPR-resistant phage.

Structure and transformation of bacteriophage A511 baseplate and tail upon infection of Listeria cells.

Contractile injection systems (bacteriophage tails, type VI secretions system, R-type pyocins, etc.) utilize a rigid tube/contractile sheath assembly for breaching the envelope of bacterial and eukaryotic cells. Among contractile injection systems, bacteriophages that infect Gram-positive bacteria represent the least understood members. Here, we describe the structure of Listeria bacteriophage A511 tail in its pre- and post-host attachment states (extended and contracted, respectively) using cryo-electron microscopy, cryo-electron tomography, and X-ray crystallography. We show that the structure of the tube-baseplate complex of A511 is similar to that of phage T4, but the A511 baseplate is decorated with different receptor-binding proteins, which undergo a large structural transformation upon host attachment and switch the symmetry of the baseplate-tail fiber assembly from threefold to sixfold. For the first time under native conditions, we show that contraction of the phage tail sheath assembly starts at the baseplate and propagates through the sheath in a domino-like motion.

Nature versus Nurture: Assessing the Impact of Strain Diversity and Pregrowth Conditions on Salmonella enterica, Escherichia coli, and Listeria Species Growth and Survival on Selected Produce Items.

Inoculation studies are important when assessing microbial survival and growth in food products. These studies typically involve the pregrowth of multiple strains of a target pathogen under a single condition; this emphasizes strain diversity. To gain a better understanding of the impacts of strain diversity ("nature") and pregrowth conditions ("nurture") on subsequent bacterial growth in foods, we assessed the growth and survival of Salmonella enterica (n = 5), Escherichia coli (n = 6), and Listeria (n = 5) inoculated onto tomatoes, precut lettuce, and cantaloupe rind, respectively. Pregrowth conditions included (i) 37°C to stationary phase (baseline), (ii) low pH, (iii) high salt, (iv) reduced water activity, (v) log phase, (vi) minimal medium, and (vii) 21°C. Inoculated tomatoes were incubated at 21°C; lettuce and cantaloupe were incubated at 7°C. Bacterial counts were assessed over three phases, including initial reduction (phase 1), change in bacterial numbers over the first 24 h of incubation (phase 2), and change over the 7-day incubation (phase 3). E. coli showed overall decline in counts (<1 log) over the 7-day period, except for a <1-log increase after pregrowth in high salt and to mid-log phase. In contrast, S. enterica and Listeria showed regrowth after an initial reduction. Pregrowth conditions had a substantial and significant effect on all three phases of S. enterica and E. coli population dynamics on inoculated produce, whereas strain did not show a significant effect. For Listeria, both pregrowth conditions and strain affected changes in phase 2 but not phases 1 and 3.IMPORTANCE Our findings suggest that inclusion of multiple pregrowth conditions in inoculation studies can best capture the range of growth and survival patterns expected for Salmonella enterica and Escherichia coli present on produce. This is particularly important for fresh and fresh-cut produce, where stress conditions encountered by pathogens prior to contamination can vary widely, making selection of a typical pregrowth condition virtually impossible. Pathogen growth and survival data generated using multiple pregrowth conditions will allow for more robust microbial risk assessments that account more accurately for uncertainty.

Microbiological quality and safety of minimally processed parsley (Petroselinum crispum) sold in food markets, southeastern Brazil.

AIMS: This study evaluated the microbiological quality and safety of minimally processed parsley sold in southeastern Brazilian food markets. METHODS AND RESULTS: One hundred samples were submitted to the enumeration of Enterobacteriaceae by plating on MacConkey agar. Colonies of Enterobacteriaceae were randomly selected and identified by MALDI-TOF MS. Samples were also tested for Listeria monocytogenes and Salmonella sp. The mean count of Enterobacteriaceae was 6·0 ± 1·0 log CFU per gram, while 18 genera (including 30 species) of bacteria belonging to this family were identified. Salmonella and L. monocytogenes were not detected, while L. innocua was found in two samples and L. fleischmannii was found in one sample. Moreover generic Escherichia coli was found in three samples, all from different brands of minimally processed parsley. CONCLUSIONS: Even though microbial pathogens were not isolated, a variety of indicator micro-organisms were identified, including vegetable spoilers and species capable of causing human opportunistic infections. These results suggest hygienic failures and/or lack of temperature control during processing and storage of these ready-to-eat products. SIGNIFICANCE AND IMPACT OF STUDY: This study highlights the need for control measures during the production chain of minimally processed parsley in order to reduce microbial contamination and the risks of foodborne diseases.

Application of an innovative water-assisted ultraviolet C light technology for the inactivation of microorganisms in tomato processing industries.

We aimed to study the efficacy of a water-assisted UVC light device (WUVC) as an innovative clean technology for the disinfection of fresh sound tomatoes and processing wash water and water turbidity was evaluated as a critical parameter. First, wash waters with different turbidities (from 0.4 to 828 NTU) were inoculated with Listeria innocua and treated in the WUVC device at different dosages. Secondly, fresh tomatoes, inoculated with L. innocua and non-inoculated ones, were treated using the WUVC device containing wash water of different turbidities for different times. The reduction of L. innocua populations on wash water and on the surface of tomato was influenced by turbidity; lower reduction values were observed at higher turbidities. Washing tomatoes with tap water with UVC lamps off (control treatment, TW) decreased L. innocua population on the surface of tomatoes but did not eliminate those bacteria that went into the water. Contrarily, when UVC lights were on, L. innocua population in wash water after treatment significantly decreased, those in clean water being the lowest populations. Reductions of native microbiota on the clean water treated with the highest UV-C radiation dose were lower than those obtained when tomatoes were artificially inoculated. We demonstrated that high reductions of L. innocua population on fresh tomatoes could be achieved using the WUVC system but some drawbacks related to the increase of turbidity should be solved for its implementation in real conditions.

Investigation of combinations of rationally selected bioengineered nisin derivatives for their ability to inhibit Listeria in broth and model food systems.

In this study, we examined the ability of nisin A and a rationally assembled bank of 36 nisin derivative producing Lactococcus lactis strains to inhibit Listeria. A broth-based bioluminescence assay for screening single and combinations of bioengineered nisin derivatives using cell-free supernatants (CFS) from nisin derivative producing strains was developed. In this way, we screened 630 combinations of nisin derivative producing strains, identifying two (CFS from M17Q + N20P and M17Q + S29E) which exhibited enhanced anti-listerial activity when used together compared to when used alone, or to the nisin A producing strain. Minimal inhibitory concentration assays performed with purified peptides revealed than when used singly, the specific activities of M17Q, N20P and S29E (3.75-7.5 µM) against L. innocua were equal to, or less than that of nisin A (MIC of 3.75 µM). Broth-based growth curve assays using purified peptides demonstrated that use of the double peptide combinations and a triple peptide combination (M17Q + N20P + S29E) resulted in an extended lag phase of L. innocua, while kill curve assays confirmed the enhanced bactericidal activity of the combinations in comparison to the single derivative peptides or nisin A. Furthermore, the enhanced activity of the M17Q + N20P combination was maintained in a model food system (frankfurter homogenate) at both chill (4 °C) and abusive (20 °C) temperature conditions, with final cell numbers significantly less (1-2 log10 CFU/ml) than those observed with the derivative peptides alone, or nisin A. To our knowledge, this study is the first investigation that combines bioengineered bacteriocins with the aim of discovering a combination with enhanced antimicrobial activity.

Antimicrobial activity of enterocins against Listeria sp. and other food spoilage bacteria.

OBJECTIVE: To determine bacteriocin producers and the prevalence of structural enterocin genes and to detect the spectrum of activity against foodborne pathogens, from isolates of Enterococcus faecium and Enterococcus faecalis that were isolated from food and the environment. RESULTS: The entA, entB, entP, ent1071 and entX genes, which encode enterocins were the most frequently observed. Enterocins were thermostable, proteinaceous, and resistant to catalase. None of the isolates produced hemolysin, and inhibition resulting from bacteriophage lysis was excluded. The bactericidal effect of enterocins against L. innocua 12612 was determined by optical density and colony forming units. For the activity spectrum, elimination of mainly Listeria strains, Bacillus sp. and clinical enterococci, was observed. Imaging with scanning electron microscopy after treatment with enterocin Efm22 showed irregular rod-shaped cells and loss of cellular integrity. CONCLUSIONS: The isolates evaluated in this study are candidates for the production of enterocins that will be used as food biopreservatives, because they have high anti-listerial activity even after 24 h of experimentation, and used in the pharmaceutical area because they inhibit clinical microorganisms.

Comparison of predicted and impedance determined growth of Listeria innocua in complex food matrices.

Indirect impedance has been used for the detection and enumeration of bacteria, however there is limited data regarding the ability of the method to measure growth and inhibition of microorganisms in food in response to preservatives. The aim of this study was to evaluate the suitability of the technique to determine maximum growth rates of Listeria innocua (used as a surrogate for Listeria monocytogenes) in complex food matrices to which multiple preservative factors had been applied and assess the suitability of the data for use in predictive microbiology. Growth of L. innocua in laboratory medium (BHI broth) and two food matrices (zucchini purée and béarnaise sauce) under varying conditions of pH (5 & 5.3), water activity (0.93, 0.96 & 0.98) and acetic and propionic acid concentration (0, 1 & 2 mM) was monitored by the conductimetric Rapid Automated Bacterial Impedance Technology (R.A.B.I.T) system by means of CO2 emission for up to 120 h. Growth rates of L. innocua were determined for several conditions across the three test matrices and a good correlation between detection times and initial inoculum level was observed in most cases (R2 ≥ 0.82). However, growth of L. innocua was not detected in a large number of conditions and comparison of growth rates determined by indirect impedance to those determined by plate counts indicated that in general, the R.A.B.I.T. system under-estimated growth. This study demonstrates that there are limitations associated with the technology, and as a result the system may be unsuitable for measuring microbial growth rates in complex food matrices under the environmental conditions tested and within the time duration of the study.

Cellulose nanofiber (CNF)-sakacin-A active material: production, characterization and application in storage trials of smoked salmon.

BACKGROUND: Sakacin-A due to its specific antimicrobial activity may represent a good candidate to develop active packaging solutions for food items supporting Listeria growth. In the present study a protein extract containing the bacteriocin sakacin-A, produced by Lactobacillus sakei Lb 706 in a low-cost culture medium containing deproteinized cheese whey, was adsorbed onto cellulose nanofibers (CNFs) to obtain an active material to be used as a mat (or a separator) in direct contact with foods. RESULTS: The applied fermentation conditions allowed 4.51 g L-1 of freeze-dried protein extract to be obtained, characterized by an antimicrobial activity of near 16 700 AU g-1 , that was used for the preparation of the active material by casting. The active material was then characterized by infrared spectra and thermogravimetric analyses. Antimicrobial trials were carried out in vitro using Listeria innocua as indicator strain; results were also confirmed in vivo, employing smoked salmon fillets intentionally inoculated with Listeria innocua: its final population was reduced to about 2.5-3 Log cycles after 28 days of storage at 6 °C in presence of sakacin-A, compared with negative control mats produced without the bacteriocin extract. CONCLUSION: This study demonstrates the possibility of producing an antimicrobial active material containing sakacin-A absorbed onto CNFs to decrease Listeria population in smoked salmon, a ready-to eat-food product. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Biotechnological Method of Preparation and Characterization of Recombinant Antimicrobial Peptide Avicin A from Enterococcus avium.

Avicin A is a bacteriocin from the gram-positive bacterium Enterococcus avium. It exhibits a high microbicidal activity against bacteria of the genus Listeria, a causative agent of the severe human infection listeriosis. We developed a biotechnological method for obtaining avicin A and characterized its structure and biological activity. We also proposed a possible mechanism of the antimicrobial action of avicin A.

Listeria monocytogenes - Danger for health safety vegetable production.

The microbiologically contaminated vegetables represent a risk for consumers, especially vegetables without thermal processing. It is known that human pathogen bacteria, such as Listeria monocytogenes, could exist on fresh vegetables. The fresh vegetables could become Listeria-contaminated if they come in touch with contaminated soil, manure, irrigation water. The aim of this work was to investigate the presence of Listeria spp. and L. monocytogenes in different kind of vegetables grown in field and greenhouse condition as well as surface and endophytic colonization plant roots of different vegetables species by L. monocytogenes in laboratory conditions. The detection of Listeria spp. and L. monocytogenes in vegetable samples was done using ISO and PCR methods. The investigation of colonization vegetable roots and detection Listeria-cells inside plant root tissue was done using Fluorescence in situ hybridization (FISH) method in combination with confocal laser scanning microscopy (CLSM). The results showed that 25.58% vegetable samples were positive for Listeria spp. and only one sample (carrot) was positive for L. monocytogenes out of 43 samples in total collected from field and greenhouse. The strain L. monocytogenes EGD-E surface and endophytic colonized carrot root in highest degree while strain L. monocytogenes SV4B was the most represented at leafy vegetable plants, such at lettuce (1.68 × 106 cells/mm3 absolutely dry root) and spinach (1.39 × 106 cells/mm3 absolutely dry root) root surface. The cells of L. monocytogenes SV4B were visible as single cells in interior tissue of plant roots (celery and sweet corn roots) as well as in the interior of the plant root cell at sweet corn root. The cells of L. monocytogenes EGD-E bind to the surface of the plant root and they were less commonly found out on root hair. In the inner layers of the root, those bacterial cells were inhabited intercellular spaces mainly as single cells very close to the larval vessels of root. Our results suggest that L. monocytogenes is very good endophytic colonizer of vegetable plant roots.

High pressure processing effect on different Listeria spp. in a commercial starter-free fresh cheese.

In this study, both microbial inactivation and growth of Listeria spp. inoculated in commercial free-starter fresh cheese was evaluated after high-pressure processing (HPP). HPP conditions (300, 400, 500 and 600 MPa at 6 °C for 5 min) and inoculum level (3-4 or 6-7 log CFU/g of cheese), as well as differences among strains inoculated (Listeria innocua, L. monocytogenes CECT 4031 and L. monocytogenes Scott A) were investigated. Inactivation and generation of sublethal injury were determined after HPP using ALOA (Agar Listeria according to Ottaviani and Agosti) and TAL (Thin Agar Layer) plating methods, respectively. Listeria inactivation increased with the pressure applied, presenting some statistical differences between the employed strains, inoculum level and sublethal injury. The highest lethality values were obtained at 600 MPa for the three strains tested, although the 500 MPa treatment presented high lethality for L. innocua and L. monocytogenes CECT 4031. After treatment, L. innocua and L. monocytogenes CECT 4031 counts in fresh cheese increased gradually during cold storage. By contrast, counts in cheeses inoculated with L. monocytogenes Scott A did not change significantly (p ≥ 0.05), being this strain the most pressure resistant and with the slowest growth rate. The manuscript present information supporting that, strains with high-level resistance should be employed during inactivation studies, instead of surrogate microorganisms. Application of HPP treatments of 500 MPa and especially 600 MPa on fresh cheeses would be effective to eliminate the most resistant microorganism to a level that should not present a public health risk under normal conditions of distribution and storage.

Predicting the combinatorial effects of water activity, pH and organic acids on Listeria growth in media and complex food matrices.

The aim of this study was to develop a model to predict growth of Listeria in complex food matrices as a function of pH, water activity and undissociated acetic and propionic acid concentration i.e. common food hurdles. Experimental growth curves of Listeria in food products and broth media were collected from ComBase, the literature and industry sources from which a bespoke secondary gamma model was constructed. Model performance was evaluated by comparing predictions to measured growth rates in growth media (BHI broth) and two adjusted food matrices (zucchini purée and béarnaise sauce). In general, observed growth rates were higher in broth than in the food matrices which resulted in the model over-estimating growth in the adjusted food matrices. In addition, model outputs were more accurate for conditions without acids, indicating that the organic acid component of the model was a source of inaccuracy. In summary, a new predictive growth model for innovating or renovating food products that rely on multi-hurdle technology was created. This study is the first to report on modelling of propionic acid as an inhibitor of Listeria in combination with other hurdles. Our findings provide valuable insights into predictive model design and performance and highlight the importance of experimental validation of models in real food matrices rather than laboratory media alone.

Selective amino acid substitution reduces cytotoxicity of the antimicrobial peptide mastoparan.

The emergence of antibiotic-resistant clinical isolates and the decreased rate of development of new antibiotics are a constant threat to human health. In this context, the therapeutic value of mastoparan (MP), a toxin from wasp venom, has been extensively studied. However, since MP shows significant cytotoxic activities, further optimization is needed. Here we evaluated the antimicrobial and cytolytic activities of an MP analog created by Ala-substitution in positions 5 and 8, named [I5, R8] mastoparan ([I5, R8] MP). We found that [I5, R8] MP displayed a broad-spectrum antimicrobial activity against bacteria and fungi (MIC in the range 3-25µM), without being hemolytic or cytotoxic toward HEK-293 cells. In addition, [I5, R8] MP-amide was highly potent (MIC=3µM) against antibiotic-resistant bacteria. The interaction with microbial membranes was investigated revealing that [I5, R8] MP is able to form an active amphipathic α-helix conformation and to disturb membranes causing lysis and cell death. Based on our findings, we hypothesize that [I5, R8] MP follows a mechanism of action similar to that proposed for MP, where the pore-forming activity leads to cell death. Our results indicate that hydrophobic moment modified by amino acid substitution may enhance MP selectivity.

Influence of application sequence and timing of eugenol and lauric arginate (LAE) on survival of spoilage organisms.

The effectiveness of sequential applications of the antimicrobials eugenol and lauric arginate (LAE) was investigated against Staphylococcus carnosus, Listeria innocua, Escherichia coli K12, and Pseudomonas fluorescens. The antimicrobials were applied simultaneously at half of their minimum lethal concentrations (MLC) or sequentially at t = 0 h and t = 3, 4, 6 or 8 h. Bacterial survival was determined by direct plate counts. Survivals kinetic were fitted to a growth and mortality model to obtain characteristic parameters that described time-dependent changes from growth to mortality or vice versa. The most effective was a simultaneous exposure of both antimicrobials to the spoilage organisms at the beginning of the incubation period. Efficiency decreases depending on order and timing of the two antimicrobials were observed upon sequential treatments. These were most effective when antimicrobials where applied within a short time period (3-4 h) and when eugenol was first applied against S. carnosus and P. fluorescens. No sequence effects were observed for L. innocua, and sequential treatments proved to be ineffective against E. coli K12. These results were attributed to cells adapting to the first applied antimicrobial. In some cases, this provided protection against the second antimicrobial rendering the overall treatment less effective.

Antibacterial Activity of Neat Chitosan Powder and Flakes.

This study investigates the antibacterial activity of neat chitosan powder and flakes against three different bacterial species, Escherichia coli, Listeria innocua and Staphylococcus aureus, which are frequent causes of food spoilage. The effect of chitosan concentration and purity, as well as the influence of temperature, ionic strength (salt) and impact of a solid physical support in the medium are examined. Results show that the antibacterial activity of neat chitosan: (i) requires partial solubilisation; (ii) can be promoted by environmental factors such as adequate temperature range, ionic strength and the presence of a solid physical support that may facilitate the attachment of bacteria; (iii) depends on bacterial species, with a sensitivity order of E. coli > L. innocua > S. aureus; and (iv) increases with chitosan concentration, up to a critical point above which this effect decreases. The latter may be due to remaining proteins in chitosan acting as nutrients for bacteria therefore limiting its antibacterial activity. These results on the direct use of chitosan powder and flakes as potential antimicrobial agents for food protection at pH values lower than the chitosan pKa (6.2-6.7) are promising.

Sakacin-A antimicrobial packaging for decreasing Listeria contamination in thin-cut meat: preliminary assessment.

BACKGROUND: Minimally processed ready-to-eat products are considered a high-risk food because of the possibility of contamination with pathogenic bacteria, including Listeria monocytogenes from the animal reservoir, and the minimal processing they undergo. In this study, a sakacin-A anti-Listeria active package was developed and tested on thin-cut veal meat slices (carpaccio). RESULTS: Enriched food-grade sakacin-A was obtained from a cell-free supernatant of a Lactobacillus sakei culture and applied (0.63 mg cm-2 ) onto the surface of polyethylene-coated paper sheets to obtain an active antimicrobial package. The coating retained antimicrobial features, indicating that the process did not affect sakacin-A functionality, as evidenced in tests carried out in vitro. Thin-cut veal meat slices inoculated with Listeria innocua (a surrogate of pathogenic L. monocytogenes) were laid on active paper sheets. After 48 h incubation at 4 °C, the Listeria population was found to be 1.5 log units lower with respect to controls (3.05 vs 4.46 log colony-forming units (CFU) g-1 ). CONCLUSION: This study demonstrates the possibility of using an antimicrobial coating containing sakacin-A to inhibit or decrease the Listeria population in ready-to-eat products, thus lowering the risk of food-related diseases. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effects of Cover Crop Species and Season on Population Dynamics of Escherichia coli and Listeria innocua in Soil.

Cover crops provide several ecosystem services, but their impact on enteric bacterial survival remains unexplored. The influence of cover cropping on foodborne pathogen indicator bacteria was assessed in five cover crop/green manure systems: cereal rye, hairy vetch, crimson clover, hairy vetch-rye and crimson clover-rye mixtures, and bare ground. Cover crop plots were inoculated with Escherichia coli and Listeria innocua in the fall of 2013 and 2014 and tilled into the soil in the spring to form green manure. Soil samples were collected and the bacteria enumerated. Time was a factor for all bacterial populations studied in all fields (P < 0.001). E. coli levels declined when soil temperatures dipped to <5°C and were detected only sporadically the following spring. L. innocua diminished somewhat but persisted, independently of season. In an organic field, the cover crop was a factor for E. coli in year 1 (P = 0.004) and for L. innocua in year 2 (P = 0.011). In year 1, E. coli levels were highest in the rye and hairy vetch-rye plots. In year 2, L. innocua levels were higher in hairy vetch-rye (P = 0.01) and hairy vetch (P = 0.03) plots than in the rye plot. Bacterial populations grew (P < 0.05) or remained the same 4 weeks after green manure incorporation, although initial reductions in L. innocua numbers were observed after tilling (P < 0.05). Green manure type was a factor only for L. innocua abundance in a transitional field (P < 0.05). Overall, the impacts of cover crops/green manures on bacterial population dynamics in soil varied, being influenced by bacterial species, time from inoculation, soil temperature, rainfall, and tillage; this reveals the need for long-term studies.

Anti-biofilm and sporicidal activity of peptides based on wheat puroindoline and barley hordoindoline proteins.

The broad-spectrum activity of antimicrobial peptides (AMPs) and low probability of development of host resistance make them excellent candidates as novel bio-control agents. A number of AMPs are found to be cationic, and a small proportion of these are tryptophan-rich. The puroindolines (PIN) are small, basic proteins found in wheat grains with proposed roles in biotic defence of seeds and seedlings. Synthetic peptides based on their unique tryptophan-rich domain (TRD) display antimicrobial properties. Bacterial endospores and biofilms are highly resistant cells, with significant implications in both medical and food industries. In this study, the cationic PIN TRD-based peptides PuroA (FPVTWRWWKWWKG-NH2 ) and Pina-M (FSVTWRWWKWWKG-NH2 ) and the related barley hordoindoline (HIN) based Hina (FPVTWRWWTWWKG-NH2 ) were tested for effects on planktonic cells and biofilms of the common human pathogens including Pseudomonas aeruginosa, Listeria monocytogenes and the non-pathogenic Listeria innocua. All peptides showed significant bactericidal activity. Further, PuroA and Pina-M at 2 × MIC prevented initial biomass attachment by 85-90% and inhibited >90% of 6-h preformed biofilms of all three organisms. However Hina, with a substitution of Lys-9 with uncharged Thr, particularly inhibited Listeria biofilms. The PIN based peptides were also tested against vegetative cells and endospores of Bacillus subtilis. The results provided evidence that these tryptophan-rich peptides could kill B. subtilis even in sporulated state, reducing the number of viable spores by 4 log units. The treated spores appeared withered under scanning electron microscopy. The results establish the potential of these tryptophan-rich peptides in controlling persistent pathogens of relevance to food industries and human health. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Influence of probiotics, included in peanut butter, on the fate of selected Salmonella and Listeria strains under simulated gastrointestinal conditions.

AIMS: This study observed the behaviour of probiotics and selected bacterial pathogens co-inoculated into peanut butter during gastrointestinal simulation. METHODS AND RESULTS: Peanut butter homogenates co-inoculated with Salmonella/Listeria strains (5 log CFU ml(-1) ) and lyophilized or cultured probiotics (9 log CFU ml(-1) ) were exposed to simulated gastrointestinal conditions for 24 h at 37°C. Sample pH, titratable acidity and pathogen populations were determined. Agar diffusion assay was performed to assess the inhibitory effect of probiotic culture supernatants with either natural (3·80 (Lactobacillus), 3·78 (Bifidobacteirum) and 5·17 (Streptococcus/Lactococcus)) or neutralized (6·0) pH. Antibacterial effect of crude bacteriocin extracts were also evaluated against the pathogens. After 24 h, samples with probiotics had lower pH and higher titratable acidity than those without probiotics. The presence of probiotics caused a significant reduction (P < 0·05) in pathogen populations. Supernatants of Bifidobacterium and Lactobacillus cultures inhibited pathogen growth; however, the elevation of pH diminished their antibacterial activities. Crude bacteriocin extracts had a strain-specific inhibitory effect only towards Listeria monocytogenes. CONCLUSION: Probiotics in 'peanut butter' survived simulated gastrointestinal conditions and inhibited the growth of Salmonella/Listeria. SIGNIFICANCE AND IMPACT OF THE STUDY: Peanut butter is a plausible carrier to deliver probiotics to improve the gastrointestinal health of children in developing countries.