Phage P100 Research Articles

Hybrid Machine Learning and Experimental Studies of Antiviral Potential of Ionic Liquids against P100, MS2, and Phi6.

Viruses are a group of widespread organisms that are often responsible for very dangerous diseases, as most of them follow a mechanism to multiply and infect their hosts as quickly as possible. Pathogen viruses also mutate regularly, with the result that measures to prevent virus transmission and recover from the disease caused are often limited. The development of new substances is very time-consuming and highly budgeted and requires the sacrifice of many living organisms. Computational chemistry methods allow faster analysis at a much lower cost and, most importantly, reduce the number of living organisms sacrificed experimentally to a minimum. Ionic liquids (ILs) are a group of chemical compounds that could potentially find a wide range of applications due to their potential virucidal activity. In our study, we conducted a complex computational analysis to predict the antiviral activity of ionic liquids against three surrogate viruses: two nonenveloped viruses, Listeria monocytogenes phage P100 and Escherichia coli phage MS2, and one enveloped virus, Pseudomonas syringae phage Phi6. Based on experimental data of toxic activity (logEC90), we assigned activity classes to 154 ILs. Prediction models were created and validated according to the Organization for Economic Co-operation and Development (OECD) recommendations using the Classification Tree method. Further, we performed an external validation of our models through virtual screening on a set of 1277 theoretically generated ionic liquids and then selected 10 active ionic liquids, which were synthesized to verify their activity against the analyzed viruses. Our study proved the effectiveness and efficiency of computational methods to predict the antiviral activity of ionic liquids. Thus, computational models are a cost-effective alternative approach compared with time-consuming experimental studies where live animals are involved.

Development of Antimicrobial Paper Coatings Containing Bacteriophages and Silver Nanoparticles for Control of Foodborne Pathogens.

In this study, a novel antimicrobial formula that incorporates Listeria bacteriophage P100 and silver nanoparticles into an alginate matrix was successfully developed. Paper coated with the antimicrobial formula inhibited the growth of Listeria monocytogenes. The effects of alginate concentration on the formation of silver nanoparticles, silver concentration on the infectivity of phages, and of low alginate concentrations on the sustained release of silver and phages were explored. The highest antimicrobial activity of the alginate-silver coating was achieved with an alginate concentration of 1%. Adding phage P100 (109 PFU/mL) into the alginate-silver coating led to a synergic effect that resulted in a 5-log reduction in L. monocytogenes. A bioactive paper was then developed by coating a base paper with the antimicrobial formula at different coating weights, followed by infrared drying. The higher coating weight was a crucial factor for the maintenance of phage infectivity throughout the coating and drying processes. Phages incorporated into the alginate matrix remained functional even after high-temperature infrared drying. Taken together, an optimized coating matrix is critical in improving the antimicrobial performance of bioactive paper as well as maintaining phage infectivity during the paper manufacturing process.

Control of Listeria monocytogenes in milk by using phage cocktail

Biocontrol applications such as using phages against the contamination of Listeria monocytogenes are promising trends in terms of reducing the use of chemical additives in food industry. The aim of this study was to determine the effectiveness of Listex P100 phage (phage P100) on different Listeria monocytogenes strains (PL2, PL3, PL9 and PL10) in pasteurized milk and broth. Survival data of L. monocytogenes were successfully described by Weibull model. Time parameter of the Weibull model was used to evaluate the phage-resistances of L. monocytogenes strains. The reduction of L. monocytogenes was greater in broth than in milk regardless of the temperature level and it was significantly higher at 30 °C than at 4 °C in both media. The reductions of L. monocytogenes strains by the phage treatment were between 2.7 to 3.4 log10 units at 30 °C and 1.4 to 2.1 log10 units at 4 °C after 4 days of incubation in broth whereas 1.9 to 2.9 log10 units and 1.0 to1.6 log10 units were observed after 4 days of incubation in milk at 30 °C and 4 °C, respectively. It was found that L. monocytogenes PL2 is the most phage-resistant strain in broth at 30 °C and at 4 °C, and in milk at 30 °C, while L. monocytogenes PL9 is the most phage-resistant L. monocytogenes strain in milk at 4 °C. This study demonstrated P100 phage could be used to control L. monocytogenes counts in milk.

Innovative hurdle system towards Listeria monocytogenes inactivation in a fermented meat sausage model - high pressure processing assisted by bacteriophage P100 and bacteriocinogenic Pediococcus acidilactici

Consumers’ quest for healthier, locally produced foods, renders the demand for these products increasingly prominent. The purpose of the present work was to evaluate the impact of a non-thermal multi-hurdle approach, which associated mild high hydrostatic pressure (HHP, 300 MPa), the bacteriophage Listex™ P100, and the pediocin PA-1 producing Pediococcus acidilactici HA 6111-2, as a novel minimal processing towards Listeria monocytogenes eradication in Alheira (a traditional fermented meat sausage from Northern Portugal).The combination of the three hurdles achieved the USDA-FSIS 5 log reduction (in accordance with the standard guidelines for ready-to-eat foods), being the only treatment to elicit the absence of L. monocytogenes immediately following processing (p < 0.05). The pair association of HHP with Listex™ P100 was unable to eliminate L. monocytogenes, whilst in the HHP-pediocin PA-1 producing P. acidilactici treated samples the eradication was delayed when compared to the three hurdles combination.In addition to the listericidal effect of the HHP-phage-lactic acid bacterium treatment, no significant differences (p > 0.05) in the pH values were observed, and the semi-quantification of the in situ biosynthesized pediocin PA-1 was documented for the first time in a fermented meat sausage model.

Practical application of bacteriophage in food manufacturing facilities for the control of Listeria sp.

AbstractListeria monocytogenes is a foodborne pathogen with the ability to persist and form biofilm matrices in processing environments of food manufacturing facilities. Bacteriophages are bacterial viruses with host specific lethality. Published research on the application of phage to control Listeria sp. in manufacturing environments is limited. In this study, we have assessed the capacity of bacteriophage P100 (Listex™) to reduce incidence of Listeria sp. in the ready‐to‐eat (RTE) environment of refrigerated (4°C) and ambient (20°C) temperature facilities using two different application strategies. A moderate application applied as a single treatment every 24 hr over three days (2 × 107 PFU/ml) and an intensified application applied once every 6 hr over a 24 hr period (1 × 108 PFU/ml). Environmental nonfood contact surface (NFCS) samples were collected and analyzed for the presence of Listeria sp. before and after treatment. When the moderate treatment protocol was applied the incidence of positives decreased from 51.3 to 17.5% in the 4°C environment and from 67.5 to 23.1% in the 20°C production area. For the intensified phage treatment method, the initial positive rate in the 4°C environment ranged from 5 to 47.5%, with an overall 43% reduction in Listeria sp. In the 20°C facility, initial environmental Listeria sp. ranged from 15 to 50%, with an overall reduction of 32% after treatment with phage P100. Data indicate the application of Listeria specific phage P100 in RTE food production environments by either the moderate or intensified application method can reduce incidence and be considered an additional intervention strategy for controlling this pathogen on NFCS.

Growth Behavior of Listeria monocytogenes in a Traditional Norwegian Fermented Fish Product (Rakfisk), and Its Inhibition through Bacteriophage Addition.

Listeria monocytogenes may persist in food production environments and cause listeriosis. In Norway, a product of concern is the traditional and popular fermented fish product “rakfisk”, which is made from freshwater salmonid fish by mild-salting and brine maturation at low temperatures for several months. It is eaten without any heat treatment, and L. monocytogenes, therefore, poses a potential hazard. We investigated the effect of salt and temperature on the growth of L. monocytogenes in rakfisk during the 91 days of maturation. The amounts of organic acids produced during fermentation were too low to inhibit growth of L. monocytogenes. Temperature was clearly the most important parameter for controlling L. monocytogenes. At 7 °C, approximately 2 log growth was observed during the first 14 days of fermentation, and the level of L. monocytogenes thereafter remained constant. At 4 °C, only a little growth potential of the pathogen was recorded. We also investigated the effect of the anti-Listeria bacteriophage P100 on rakfisk with added L. monocytogenes. The phage was introduced to the L. monocytogenes-inoculated fish before fermentation, and an average of 0.9 log reduction was observed throughout the fermentation period. This is the first study of L. monocytogenes behavior in rakfisk and points to possible measures for increasing the product safety.

Non-thermal approach to Listeria monocytogenes inactivation in milk: The combined effect of high pressure, pediocin PA-1 and bacteriophage P100

Non-thermal food processing and replacement of chemical additives by natural antimicrobials are promising trends in the food industry. The objective of the present work was to evaluate the effect of a process which combines mild high hydrostatic pressure – HHP (200 and 300 MPa, 5 min, 10 °C), phage Listex™ P100 and the bacteriocin pediocin PA-1 as a new non-thermal process for destruction of Listeria monocytogenes (104 CFU mL−1 or 107 CFU mL−1) in milk. For inoculum levels of 104 CFU mL−1, HHP combined with phage P100 eliminated L. monocytogenes immediately after pressurization. When L. monocytogenes was inoculated at levels of 107 CFU mL−1, a synergistic effect between phage P100, pediocin PA-1 and HHP (300 MPa) on the inactivation of L. monocytogenes was observed during storage of milk at 4 °C. For non-pressure treated samples inoculated with phage or pediocin or both, L. monocytogenes counts decreased immediately after biocontrol application, but regrowth was observed in a few samples during storage. Phage particles were stable during refrigerated storage for seven days while pediocin PA-1 remained stable only during three days. Further studies will have to be performed to validate the findings of this work in specific applications (e.g. production of raw milk cheese).

Effects of Bacteriophage P100 at Different Concentrations on the Structural Parameters of Listeria monocytogenes Biofilms

Because listeriosis is one of the deadliest foodborne diseases, controlling and eradicating Listeria monocytogenes biofilms is a serious challenge for food safety. Biofilms (24 h old) formed on polystyrene by a L. monocytogenes strain of food origin were exposed for a further 24 h to 12 different concentrations (from 100 to 1011 PFU/mL) of the bacteriophage P100 (Listex P100). The structural parameters of biofilms were studied by using confocal laser scanning microscopy and digital image analysis. The biovolume in the observation field (14,121 μm2) of control (untreated) biofilms was 237,333.1 ± 2,692.6 μm3. The biomass of treated biofilms ranged from 164.7 ± 89.0 μm3 (biofilms exposed to 1010 PFU/mL) to 231,170.5 ± 15,142.0 μm3 (100 PFU/mL). The lowest biomass was achieved after treatment with 108 PFU/mL, with no further decrease in biovolume when higher phage concentrations were used. A strong ( P < 0.001) correlation was found between phage concentration (log units) and biovolume (-0.965), surface coverage (-0.939), roughness (0.976), maximum thickness (-0.853), and average thickness (-0.965). Findings from this research suggest that bacteriophage P100 at concentrations equal to or greater than 8 log PFU/mL successfully removes L. monocytogenes biofilms from polystyrene surfaces.

The protective effect of food matrices on Listeria lytic bacteriophage P100 application towards high pressure processing

The application of lytic phages as biocontrol agents is emerging as a promising strategy towards elimination or reduction of foodborne pathogens in a variety of food products. This technology is particularly advantageous for minimally processed and ready-to-eat (RTE) foods. In this study, the potential use of Listex™ P100 combined with high hydrostatic pressure (HPP), to enhance the control of Listeria monocytogenes in food was evaluated. For that, the effect of three pressures (200, 300 or 400 MPa; 5 min, 10 °C) on phage P100 stability was tested when inoculated in six different matrices: phosphate buffered saline (PBS, pH 7.4); apple juice (pH 3.41); orange/carrot nectar (pH 3.54); UHT whole milk (pH 6.73); and, two traditional Portuguese fermented products, “Serra da Estrela” cheese (pH 5.66) and “Alheira”, a meat sausage (pH 6.07). The results showed that treatment at 400 MPa reduced phage titres to below the detection level in all matrices, whereas at milder pressures the survival of the phage was matrix dependent. “Alheira”, “Serra da Estrela” cheese and UHT whole milk were shown to be baroprotective matrices that support phage P100 application in HHP up to 300 MPa; however, an accentuated phage inactivation was observed in apple and orange/carrot nectar, which may be related to the acidic pH values of these matrices. The initial phage load did not affect the inactivation rate during HHP processing (300 MPa, 5 min, 10 °C) in PBS, cheese, sausage or milk matrices, and the phage titres were stable in these matrices during storage at 4 °C for 28 days for milk and 60 days for “Alheira” and “Serra da Estrela” cheese. In addition, a baroprotective effect on phage stability was observed when PBS was supplemented with reducing sugars, dextrin, casein, and tween 80. In conclusion, at mild HHP treatment, phage P100 remained active in specific matrices and seems to present potential to be added in non-thermal inactivation of L. monocytogenes.

Efficacy of LISTEX P100 at Different Concentrations for Reduction of Listeria monocytogenes Inoculated in Sashimi

Sushi restaurants have become quite popular in Europe, with an increase in the consumption of the sashimi speciality. Pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus have been reported in this kind of food. Controlling the presence and multiplication of L. monocytogenes is a challenge for food safety management systems owing to its ubiquitous presence and psychrotrophic growth. Bacteriophages have been used as pathogenic biocide agents for decades. The bacteriophage P100, present in LISTEX P100, was used in this study to understand the possibility of implementing a new critical control point for L. monocytogenes in sashimi preparation and sale. Different concentrations of LISTEX P100 were used for reduction of L. monocytogenes in inoculated samples at 3 and 22°C. The reduction in initial counts of 2 log CFU/g was effective in the first 24 h with the 8-log PFU/g inoculation. Promising results were obtained in assays with the 6-log CFU/g initial counts and the 8-log PFU/g inoculation, at 22°C, achieving a maximum reduction of 4.44 log CFU/g, compared with the control group. These results seem to confirm that bacteriophages can be an option in reducing the population of the L. monocytogenes pathogenic bacteria in sashimi, mainly in takeaway sales.

Virucidal Influence of Ionic Liquids on Phages P100 and MS2.

An increasing number of publications describe the potential of ionic liquids (ILs) as novel antimicrobials, antibacterial coatings and even as active pharmaceutical ingredients. Nevertheless, a major research area, notably their impact on viruses, has so far been neglected. Consequently the aim of this study was to examine the effects of ILs on the infectivity of viruses. A systematic analysis to investigate the effects of defined structural elements of ILs on virus activity was performed using 55 ILs. All structure activity relationships (SARs) were tested on the human norovirus surrogate phage MS2 and phage P100 representing non-enveloped DNA viruses. Results demonstrate that IL SAR conclusions, established for prokaryotes and eukaryotes, are not readily applicable to the examined phages. A virus-type-dependent IL influence was also apparent. Overall, four ILs, covering different structural elements, were found to reduce phage P100 infectivity by ≥4 log10 units, indicating a virucidal effect, whereas the highest reduction for phage MS2 was about 3 log10 units. Results indicate that future applications of ILs as virucidal agents will require development of novel SARs and the obtained results serve as a good starting point for future studies.

Influence of Environmental Factors on Phage-Bacteria Interaction and on the Efficacy and Infectivity of Phage P100.

When using bacteriophages to control food-borne bacteria in food production plants and processed food, it is crucial to consider that environmental conditions influence their stability. These conditions can also affect the physiological state of bacteria and consequently host–virus interaction and the effectiveness of the phage ability to reduce bacteria numbers. In this study we investigated the stability, binding, and replication capability of phage P100 and its efficacy to control Listeria monocytogenes under conditions typically encountered in dairy plants. The influences of SDS, Lutensol AO 7, salt, smear water, and different temperatures were investigated. Results indicate that phage P100 is stable and able to bind to the host under most conditions tested. Replication was dependent upon the growth of L. monocytogenes and efficacy was higher when bacterial growth was reduced by certain environmental conditions. In long-term experiments at different temperatures phages were initially able to reduce bacteria up to seven log10 units after 2 weeks at 4°C. However, thereafter, re-growth and development of phage-resistant L. monocytogenes isolates were encountered.

Phage Inactivation of Listeria monocytogenes on San Daniele Dry-Cured Ham and Elimination of Biofilms from Equipment and Working Environments.

The anti-listerial activity of generally recognized as safe (GRAS) bacteriophage Listex P100 (phage P100) was demonstrated in broths and on the surface of slices of dry-cured ham against 5 strains or serotypes (i.e., Scott A, 1/2a, 1/2b, and 4b) of Listeria monocytogenes. In a broth model system, phage P100 at a concentration equal to or greater than 7 log PFU/mL completely inhibited 2 log CFU/cm2 or 3 log CFU/cm2 of L. monocytogenes growth at 30 °C. The temperature (4, 10, 20 °C) seemed to influence P100 activity; the best results were obtained at 4 °C. On dry-cured ham slices, a P100 concentration ranging from 5 to 8 log PFU/cm2 was required to obtain a significant reduction in L. monocytogenes. At 4, 10, and 20 °C, an inoculum of 8 log PFU/cm2 was required to completely eliminate 2 log L. monocytogenes/cm2 and to reach the absence in 25 g product according to USA food law. Conversely, it was impossible to completely eradicate L. monocytogenes with an inoculum of approximately of 3.0 and 4.0 log CFU/cm2 and with a P100 inoculum ranging from 1 to 7 log PFU/cm2. P100 remained stable on dry-cured ham slices over a 14-day storage period, with only a marginal loss of 0.2 log PFU/cm2 from an initial phage treatment of approximately 8 log PFU/cm2. Moreover, phage P100 eliminated free L. monocytogenes cells and biofilms on the machinery surfaces used for dry-cured ham production. These findings demonstrate that the GRAS bacteriophage Listex P100 at level of 8 log PFU/cm2 is listericidal and useful for reducing the L. monocytogenes concentration or eradicating the bacteria from dry-cured ham.

Biocontrol of Listeria monocytogenes in fish by enterocin AS-48 and Listeria lytic bacteriophage P100

The purpose of this study was to determine the ability of enterocin AS-48 to control Listeria monocytogenes in fish during storage at 4 °C. AS-48 (0.37 μg/cm2) was tested singly and in combination with phage P100 (2.3 × 107 PFU/cm2) on a cocktail of 103 CFU/cm2 L. monocytogenes strains inoculated in fillet tissues of raw hake and salmon and in smoked salmon. In raw fish, AS-48 alone reduced listeria with respect to the untreated control by 1.68, 2.79, 2.9, and 3.13 log CFU/cm2 (in hake) and by 1.9, 2.55, 2.8, and 2.8 (in salmon) at 1, 2, 3, and 7 d, respectively. Phage P100 treatment also yields significant reductions (but lower than AS-48) in listeria counts in both raw fishes. A combined treatment of AS-48/P100 eliminated listeria from hake and salmon fillets from 2 and 1 days respectively. In smoked salmon, AS-48 reduced listeria by 2, 3.4, 4.5, 4.25, and 4.25 log CFU/cm2 with respect to the control at 1, 5, 10, 15, and 30 d, respectively. P100 treatment also reduced listeria counts but in lesser quantities than AS-48. A combined treatment of AS-48/P100 reduced listeria below detection levels from 1 to 15 d; afterwards, a slight listeria reactivation was detected.

Screening and characterisation of bacteriophage P100 insensitive Listeria monocytogenes isolates in Austrian dairy plants

Listeria monocytogenes is a major health threat in food production premises. The use of bacteriophages, such as listeriaphage P100, to minimize food pathogens is a promising alternative to eliminate bacteria, as is application of Listex™ P100. However, the use of phages may result in resistant L. monocytogenes strains. In this study 486 L. monocytogenes isolates obtained from 59 dairies over 15 years were screened for the presence of P100 insensitive L. monocytogenes. The overall number of P100 insensitive L. monocytogenes isolates was 13 (2.7%) for all years and all investigated plants. Insensitivities were detected in five of 59 dairies. Detected insensitive isolates did not appear randomly, but were in connection with phage treatments.The influence of dilution on the efficacy of P100 was tested by different host pathogen ratios. Additionally, artificially induced insensitive isolates were selected. Adsorption tests suggested that the detected insensitive L. monocytogenes isolates had receptor modifications.

Reduction of Listeria monocytogenes in cold‐smoked salmon by bacteriophage P100, nisin and lauric arginate, singly or in combinations

SummaryThree GRAS antimicrobials including, lauric arginate (LAE), bacteriophage P100 (phage P100) and bacteriocin nisin, were evaluated either singly or in combinations for the reduction of initial load of Listeria monocytogenes in cold‐smoked salmon (CSS). The stability of phage P100 in the presence of LAE (200 ppm) and nisin (500 ppm) or at 10× and 100× of these concentrations was determined at 4 °C or 30 °C for 24 h in a broth model. Phage P100 was found to be highly stable in the presence of these antimicrobial agents as plaque‐forming units (PFU) did not vary between control and antimicrobial‐treated phage. The survival of L. monocytogenes in the presence of phage P100, nisin and LAE showed remarkable reduction within 24 h both at 4 °C or 30 °C in broth. Treatment of CSS containing 3.5 log CFU cm−2 L. monocytogenes with phage P100 (108 PFU mL−1), nisin (500 ppm) and LAE (200 ppm) showed strong listericidal action and reduced the L. monocytogenes by 2–3 log CFU cm−2 after 24 h. Among the combined treatments, phage P100 + LAE or nisin + LAE exhibited the most listericidal action in which L. monocytogenes cells were reduced to undetectable level within 24 h in CSS.

Control of Listeria monocytogenes growth in soft cheeses by bacteriophage P100.

The purpose of this study was to determine the effect of bacteriophage P100 on strains of Listeria monocytogenes in artificially inoculated soft cheeses. A mix of L. monocytogenes 1/2a and Scott A was inoculated in Minas Frescal and Coalho cheeses (approximately 10(5) cfu/g) with the bacteriophage added thereafter (8.3 × 10(7) PFU/g). Samples were analyzed immediately, and then stored at 10 °C for seven days. At time zero, 30 min post-infection, the bacteriophage P100 reduced L. monocytogenes counts by 2.3 log units in Minas Frescal cheese and by 2.1 log units in Coalho cheese, compared to controls without bacteriophage. However, in samples stored under refrigeration for seven days, the bacteriophage P100 was only weakly antilisterial, with the lowest decimal reduction (DR) for the cheeses: 1.0 log unit for Minas Frescal and 0.8 log units for Coalho cheese. The treatment produced a statistically significant decrease in the counts of viable cells (p < 0.05) and in all assays performed, we observed an increase of approximately one log cycle in the number of viable cells of L. monocytogenes in the samples under refrigeration for seven days. Moreover, a smaller effect of phages was observed. These results, along with other published data, indicate that the effectiveness of the phage treatment depends on the initial concentration of L. monocytogenes, and that a high concentration of phages per unit area is required to ensure sustained inactivation of target pathogens on food surfaces.

Reduction of Listeria monocytogenes in queso fresco cheese by a combination of listericidal and listeriostatic GRAS antimicrobials

Single and combined effects of three GRAS (generally recognized as safe) antimicrobials including, bacteriophage P100 (phage P100), lauric arginate (LAE), and potassium lactate–sodium diacetate mixture (PL–SD) were evaluated against Listeria monocytogenes cold growth in queso fresco cheese (QFC). The fate of phage P100 when exposed to LAE (200ppm) or PL–SD (2.8% PL and 0.2% SD) was determined at 4°C and 30°C in a broth model. Phage P100 was found to be stable in the presence of these antimicrobial agents as plaque forming units (PFU) did not vary between control, LAE or PL–SD treatments. When 9logCFU/ml of stationary phase cells of L. monocytogenes was exposed to these antimicrobials in tryptic soy broth, there was a 3 to 5logCFU/ml reduction with phage P100 and a complete 9logCFU/ml reduction with LAE but no measurable reduction with PL–SD after 24h at 4°C or 30°C. In QFC, the L. monocytogenes populations increased from the initial 3.5logCFU/cm2 to 7.7logCFU/cm2 in 28days at 4°C. Treatment with 7.8logPFU/cm2 of phage P100 or 200ppm of LAE showed strong listericidal effect initially by reducing L. monocytogenes counts by 2 to 3.5–4logCFU/cm2 while there was a subsequent regrowth of L. monocytogenes at 4°C. Treatment with PL–SD showed strong listeriostatic effect without decreasing L. monocytogenes counts but growth was prevented for 28days at 4°C. Only the combined treatment of listericidal phage P100 or LAE with listeriostatic PL–SD reduced the initial L. monocytogenes counts by 2–4logCFU/cm2 and also kept the L. monocytogenes counts at that reduced level in QFC for 28days at 4°C.

Use of epifluorescence microscopy to assess the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces

One source of foodborne listeriosis is related to a virulent strain established in the food-processing environment. We used direct epifluorescence microscopy (DEM) to evaluate the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces, in wet conditions at room temperature. Biofilms that were allowed to develop for 72 h were subsequently treated with different concentrations of phage (5, 6, 7, and 8 log PFU/ml). L. monocytogenes was monitored up to 48 h, using both DEM and cultivation method. When L. monocytogenes was monitored using a cultivation method, the first significant reduction was observed after phage treatment with 7 and 8 log PFU/ml at 8 h. Subsequently, these treatments achieved undetectable levels of pathogen at 48 h, with a mean reduction of 5.29 log CFU/cm2. Conversely, when samples were evaluated using DEM in treatments with 6, 7, and 8 log PFU/ml, although disaggregation of biofilms could be observed after 8 h, viable cells were still present up to 48 h (maximal reduction 1.5 log units). The phage titer remained stable or increased up to 2.59 log units during the study period. In conclusion, phage P100 may provide an adjuvant measure to control L. monocytogenes biofilm on stainless steel surfaces. However, phage treatment must be used in combination with other hygienization measures to increase efficacy. In this study, DEM was a good tool to quickly and accurately assess the real effect of phage P100 on L. monocytogenes biofilms.

Global Transcriptional Analysis of Spontaneous Sakacin P-Resistant Mutant Strains of Listeria monocytogenes during Growth on Different Sugars

Subclass IIa bacteriocins have strong antilisterial activity and can control the growth of Listeria monocytogenes in food. However, L. monocytogenes may develop resistance towards such bacteriocins. In this follow-up study, the transcriptomes of a high level (L502-1) and a low level (L502-6) spontaneous sakacin P-resistant mutant strain of L. monocytogenes were compared to the wild-type (L502). The growth of the resistant strains was reduced on mannose but not affected on cellobiose and the transcriptomics was performed during growth on these sugars. The mannose phosphotransferase system (PTS) encoded by the mptACD operon (mpt) is known for transporting mannose and also act as a receptor to class IIa bacteriocins. The mpt was repressed in L502-1 and this is in accordance with abolition of the bacteriocin receptor with resistance to class IIa bacteriocins. In contrast, the mpt was induced in L502-6. Despite the induction of the mpt, L502-6 showed 1,000 times more resistance phenotype and reduced growth on mannose suggesting the mannose-PTS may not be functional in L502-6. The microarray data suggests the presence of other transcriptional responses that may be linked to the sakacin P resistance phenotype particularly in L502-6. Most of commonly regulated genes encode proteins involved in transport and energy metabolism. The resistant strains displayed shift in general carbon catabolite control possibly mediated by the mpt. Our data suggest that the resistant strains may have a reduced virulence potential. Growth sugar- and mutant-specific responses were also revealed. The two resistant strains also displayed difference in stability of the sakacin P resistance phenotype, growth in the presence of both the lytic bacteriophage P100 and activated charcoal. Taken together, the present study showed that a single time exposure to the class IIa bacteriocin sakacin P may elicit contrasting phenotypic and transcriptome responses in L. monocytogenes possibly through regulation of the mpt.