Selection of mutant Listeria phages under food-relevant conditions can enhance application potential.

Bacteriophages are viruses that infect and kill bacteria. Currently, phage products are available for the control of the pathogen Listeria monocytogenes in food products in the United States. In this study, we explore whether experimental evolution can be used to generate phages with improved abilities to function under specific food-relevant conditions. Ultra-pasteurized oat and whole milk were chosen as test matrices as they represent different food groups, yet have similar physical traits and macronutrient composition. We showed that (i) wild-type phage LP-125 infection kinetics are different in the two matrices and (ii) LP-125 has a significantly higher burst size in oat milk. From this, we attempted to evolve LP-125 to have improved infection kinetics in whole milk. Ancestral LP-125 was passaged through 10 rounds of amplification in milk conditions. Plaque-purified DNA samples from milk-selected phages were isolated and sequenced, and mutations present in the isolated phages were identified. We found two nonsynonymous substitutions in LP125_108 and LP125_112 genes, which encode putative baseplate-associated glycerophosphoryl diester phosphodiesterase and baseplate protein, respectively. Protein structural modeling showed that the substituted amino acids in the mutant phages are predicted to localize to surface-exposed helices on the corresponding structures, which might affect the surface charge of proteins and their interaction with the bacterial cell. The phage containing the LP125_112 mutation adsorbed significantly faster than the ancestral phage in both oat and whole milk. Follow-up experiments suggest that fat content may be a key factor for the expression of the phenotype of this mutation. IMPORTANCE Bacteriophages are one of the tools available to control the foodborne pathogen, Listeria monocytogenes. Phage products must work under a broad range of food conditions to be an effective control for L. monocytogenes. Here, we show that the experimental evolution of phages can be used to generate new phages with phenotypes useful under specific conditions. We used this approach to select for a mutant phage that more efficiently binds to L. monocytogenes that is grown in whole milk and oat milk. We show that the fat content of these milks is necessary for the expression of this phenotype. Our findings show that experimental evolution can be used to select for improved phages with better performance under specific conditions. This approach has the potential to support the development of condition-specific phage-based biocontrols in the food industry.

Control of residual phage in the evaluation of phage-based food safety applications.

Bacteriophage ("Phage") products are gaining interest in controlling foodborne pathogens as they are natural, specific, and can replicate at the site of contamination. One challenge in determining the efficacy of phage biocontrol is accounting for residual phages that may impact the recovery and the enumeration of surviving bacteria downstream from the treatment on food surface (FS) or food contact surface (FCS). Typically, the efficacy of a phage formulation is tested by applying it to a FS or FCS that has been pre-inoculated with the target pathogen and incubating the treatment for a set period of time. The food sample is transferred into a liquid buffer and stomached to release the surviving bacteria for their enumeration. During these final steps, there is a potential for residual phage to interact with bacterial survivors, which could affect the calculated efficacy of the phage. Limited studies demonstrated that bacterial reductions in these experiments occur specifically during treatment and not during sample recovery. This review highlights the importance of including appropriate controls to determine if residual phages are impacting bacterial recovery and the methods used to mitigate those impacts, which involves either neutralizing residual phages or separating residual phages from the surviving bacteria.

Control of Salmonella in low-moisture foods: Enterococcus faecium NRRL B-2354 as a surrogate for thermal and non-thermal validation.

Salmonella has been implicated in multiple foodborne outbreaks and recalls associated with low water activity foods (LawF). To verify the effectiveness of a process against Salmonella in LawF, validation using a nonpathogenic surrogate strain is essential. Enterococcus faecium NRRL B-2354 strain has been used as a potential surrogate of Salmonella in different processing of LawF. However, the survival of Salmonella and E. faecium in LawF during food processing is a dynamic function of aw, food composition and structure, processing techniques, and other factors. This review assessed pertinent literature on the thermal and non-thermal inactivation of Salmonella and its presumable surrogate E. faecium in various LawF and provided an overview of its suitibility in different LawF. Overall, based on the D-values, survival/reduction, temperature/time to obtain 4 or 5-log reductions, most studies concluded that E. faecium is a suitable surrogate of Salmonella during LawF processing as its magnitude of resistance was slightly greater or equal (i.e., statistical similar) as compared to Salmonella. Studies also showed its unsuitability which either does not provide a proper margin of safety or being overly resistant and may compromise the quality and organoleptic properties of food. This review provides useful information and guidance for future validation studies of LawF.

Assessment and Classification of Volatile Profiles in Melon Breeding Lines Using Headspace Solid-Phase Microextraction Coupled with Gas Chromatography-Mass Spectrometry.

Cucumis melo L is one of the most commercial and economical crops in the world with several health beneficial compounds as such carotenoids, amino acids, vitamin A and C, minerals, and dietary fiber. Evaluation of the volatile organic compounds (VOCs) in different melon (Cucumis melo L.) breeding lines provides useful information for improving fruit flavor, aroma, and antimicrobial levels. In this study, the VOCs in 28 melon breeding lines harvested in 2019 were identified and characterized using head space solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). This identified 113 VOCs with significant differences in composition and contents of among the breeding lines, including 15 esters, 27 aldehydes, 35 alcohols, 14 ketones, 4 acids, 10 hydrocarbons, 5 sulfurs, and 3 other compounds. The highest average contents of all the VOCs were found in BL-30 (13,973.07 µg/kg FW) and the lowest were in BL-22 (3947.13 µg/kg FW). BL-9 had high levels of carotenoid-derived VOCs. The compounds with the highest contents were benzaldehyde, geranylacetone, and ß-ionone. Quality parameters such as color and sugar contents of melons were also measured. All the melon color readings were within the typical acceptable range. BL-22 and BL-14 had the highest and lowest sugar contents, respectively. Principal component analysis (PCA) produced diverse clusters of breeding lines based on flavor and aroma. BL-4, BL-7, BL-12, BL-20, and BL-30 were thus selected as important breeding lines based on their organoleptic, antimicrobial, and health-beneficial properties.

Decreased biofilm formation by planktonic cells of Listeria monocytogenes in the presence of sodium hypochlorite.

The objective of this study was to determine if the adaptation at planktonic stage to subinhibitory concentrations (SIC) of sodium hypochlorite (NaOCl) could modulate the biofilm forming ability of five Listeria monocytogenes strains V7, Scott A, FSL-N1-227, FSL F6-154 and ATCC 19116 representing serotypes 1/2a, 4b and 4c. Biofilm formation by NaOCl nonadapted and adapted L. monocytogenes planktonic cells was measured in the presence or absence of SIC of NaOCl. The biofilm formation ability of NaOCl nonadapted and adapted L. monocyotgenes planktonic cells was reduced only in the presence of NaOCl (P < 0.05). Scanning electron microscopy revealed that the continuous exposure of NaOCl induced morphological changes in the L. monocytogenes biofilm structure and reduced its attachment to polystyrene surface. The qRT-PCR results also showed that the subinhibitory NaOCl reduced biofilm formation related gene expression such as motility and quorum sensing signals (P < 0.05). These findings indicate that subinhibitory NaOCl can reduce the ability of L. monocytogenes planktonic cells to form biofilms on polystyrene surface.

Verification of peroxyacetic acid treatment against L. monocytogenes on fresh apples using E. faecium NRRL B-2354 as a surrogate in commercial spray-bar operations.

Peroxyacetic acid (PAA) is a commonly used antimicrobial in apple spray bar interventions during post-harvest packing. However, limited information is available about its efficacy against foodborne pathogens on fresh apples under commercial packing conditions. In this study, the practical efficacies of PAA against Listeria monocytogenes on fresh apples during spray bar operation at ambient and elevated temperature were validated in three commercial packing facilities using Enterococcus faecium NRRL B-2354 as a surrogate strain. Apples were inoculated with E. faecium at ~6.5 Log10 CFU/apple and subjected to PAA spray bar interventions per commercial packing line practice. At each temperature and contact time intervention combination, 20-24 inoculated apples were processed together with 72-80 non-inoculated apples. Applying 80 ppm PAA at ambient temperature (17-21 °C) achieved a similar log reduction (P > 0.05) of E. faecium on Granny Smith apples (GSA) in three apple packing facilities, which caused 1.12-1.23 and 1.18-1.32 Log10 CFU/apple reductions of E. faecium on GSA for 30-sec and 60-sec intervention, respectively. Increasing the temperature of the PAA solution to 43-45 °C enhanced its bactericidal effect against E. faecium, causing 1.45, 1.86 and 2.19 Log10 CFU/apple reductions in three packing facilities for a 30-sec contact, and 1.50, 2.24, and 2.29 Log10 CFU/apple reductions for a 60-sec contact, respectively. Similar efficacies (P > 0.05) of PAA at both ambient and elevated temperature were also observed on Fuji apples. Spraying PAA on apples at ambient or elevated temperature reduced the level of E. faecium cross-contamination from inoculated apples to non-inoculated apples but could not eliminate cross-contamination. Data from this study provides valuable technical information and a reference point for the apple industry in controlling L. monocytogenes and verifying the effectiveness of their practices.

Salmonella enterica growth and biofilm formation in flesh and peel cantaloupe extracts on four food-contact surfaces.

Salmonella enterica is responsible for the highest number of foodborne disease outbreaks pertaining to cantaloupe industry. The objective of this study was to examine the growth and biofilm formation by outbreak strains of S. enterica ser. Poona (S. Poona), S. enterica ser. Stanley (S. Stanley) and S. enterica ser. Montevideo (S. Montevideo) on different food-contact processing surfaces in cantaloupe flesh and peel extracts at 22 °C and 10 °C. The generation time of all S. enterica strains tested was shorter in the high concentration (50 mg/ml) of cantaloupe extract and high temperature. In 50 mg/ml of cantaloupe flesh or peel extract, the populations of S. enterica were increased by 5 log CFU/ml in 24 h at 22 °C and 1 log CFU/ml in 72 h at 10 °C. In 2 mg/ml of cantaloupe flesh or peel extracts, the populations of S. enterica were increased by 3.5 log CFU/ml in 56 h at 22 °C, but there were no changes in 72 h at 10 °C. The biofilm production of S. enterica was greater at 50 mg/ml of cantaloupe extract and 22 °C, but no major differences (P ≥ 0.05) were found among the strains tested. In 50 mg/ml cantaloupe extract, S. enterica produced 5-6 log CFU/cm2 biofilm in 4-7 d at 22 °C and approximately 3.5-4 log CFU/cm2 in 7 d at 10 °C. In 2 mg/ml of cantaloupe extract, S. enterica produced 4-4.5 log CFU/cm2 biofilms in 4-7 d at 22 °C and 3 log CFU/cm2 in 7 d at 10 °C. Biofilm formation by S. Poona (01A4754) was lowest on buna-n rubber compared to stainless steel, polyethylene and polyurethane surfaces under the majority of conditions tested. Overall, these findings show that S. enterica strains can grow rapidly and form biofilms on different cantaloupe processing surfaces in the presence of low concentrations of cantaloupe flesh or peel extracts.

Scanning electron microscopy of Salmonella biofilms on various food-contact surfaces in catfish mucus.

The objective of this study was to determine the growth and survival of Salmonella enterica in the presence of high and low concentrations (375 µg/ml and 15 µg/ml) of catfish mucus extract at 10 °C and 22 °C for 63 days. The second objective of this study was to investigate the biofilm formation of Salmonella enterica serovar Blockley (7175) in catfish mucus extract for 48 h at 22 °C on four food-contact surfaces and to observe the biofilm populations using Scanning Electron Microscopy (SEM). The surface properties, surface roughness and surface energies were determined using contact angle measurement and atomic force microscopy. In 375 µg/ml of catfish mucus extract that was inoculated with 3 log CFU/ml, the growth of Salmonella counts were increased to a maximum of 6-7 log CFU/ml at 10 °C and 7-8 log CFU/ml at 22 °C in 7-14 d and decreased by 1-2 log CFU/ml from these peak levels at both 10 °C and 22 °C from 21 to 63 d. In 15 µg/ml of catfish mucus extract, Salmonella counts were in the range of 4-5 log CFU/ml at 10 °C and 5-6 log CFU/ml at 22 °C over 7-63 d of storage. By contrast, Salmonella counts were non-detectable in the absence of catfish mucus by 21-28 d of storage at 10 °C or 22 °C. The biofilm counts of S. Blockley (7175) on a stainless steel surface were 4 log CFU/cm2 and 5.5 log CFU/cm2 in 15 µg/ml and 375 µg/ml of catfish mucus extract respectively after 48 h incubation at 22 °C. SEM revelead that biofilm formation by S. Blockley (7175) was less in 15 µg/ml than 375 µg/ml of catfish mucus extract on stainless steel. In addition, SEM indicated that the visible biofilms were least on buna-N rubber as compared to stainless steel, polyethylene and polyurethane surfaces. Contact angle and atomic force microscopy confirmed that buna-N rubber was highly hydrophobic with low surface energy and low roughness when compared to other three surfaces. These findings indicate that Salmonella can utilize catfish mucus as a nutrient source to survive for longer periods and promote biofilm formation for its persistence on different food-contact surfaces.

Growth and Biofilm Formation by Listeria monocytogenes in Catfish Mucus Extract on Four Food Contact Surfaces at 22 and 10°C and Their Reduction by Commercial Disinfectants.

The objective of this study was to determine the effect of strain and temperature on growth and biofilm formation by Listeria monocytogenes in high and low concentrations of catfish mucus extract on various food contact surfaces at 10 and 22°C. The second objective of this study was to evaluate the efficacy of disinfectants at recommended concentrations and contact times for removing L. monocytogenes biofilm cells from a stainless steel surface covered with catfish mucus extract. Growth and biofilm formation of all L. monocytogenes strains increased with higher concentrations of catfish mucus extract at both 10 and 22°C. When 15 µg/mL catfish mucus extract was added to 3 log CFU/mL L. monocytogenes, the biofilm levels of L. monocytogenes on stainless steel reached 4 to 5 log CFU per coupon at 10°C and 5 to 6 log CFU per coupon at 22°C in 7 days. With 375 µg/mL catfish mucus extract, the biofilm levels of L. monocytogenes on stainless steel reached 5 to 6 log CFU per coupon at 10°C and 6 to 7.5 log CFU per coupon at 22°C in 7 days. No differences ( P > 0.05) were observed between L. monocytogenes strains tested for biofilm formation in catfish mucus extract on the stainless steel surface. The biofilm formation by L. monocytogenes catfish isolate HCC23 was lower on Buna-N rubber than on stainless steel, polyethylene, and polyurethane surfaces in the presence of catfish mucus extract ( P < 0.05). Contact angle analysis and atomic force microscopy confirmed that Buna-N rubber was highly hydrophobic, with lower surface energy and less roughness than the other three surfaces. The complete reduction of L. monocytogenes biofilm cells was achieved on the stainless steel coupons with a mixture of disinfectants, such as quaternary ammonium compounds with hydrogen peroxide or peracetic acid with hydrogen peroxide and octanoic acid at 25 or 50% of the recommended concentration, in 1 or 3 min compared with use of the quaternary ammonium compounds, chlorine, or acid disinfectants alone, which were ineffective for removing all the L. monocytogenes biofilm cells.

Biofilm formation by Salmonella spp. in catfish mucus extract under industrial conditions.

The objective of this study was to determine the effect of strain and temperature on the growth and biofilm formation of Salmonella spp. in high and low concentrations of catfish mucus extract on different food-contact surfaces at 22 °C and 10 °C. The second objective of this study was to evaluate the efficacy of disinfectants at recommended concentrations and contact times for removing Salmonella biofilms cells on a stainless steel surface containing catfish mucus extract. Growth and biofilm formation of all Salmonella strains increased with higher concentrations of catfish mucus extract at both 10 °C and 22 °C. In 15 µg/ml of catfish mucus extract inoculated with 3 log CFU/ml, the biofilm levels of Salmonella on stainless steel surface reached to 3.5 log CFU/cm2 at 10 °C or 5.5 log CFU/cm2 at 22 °C in 7 days. In 375 µg/ml of catfish mucus extract inoculated with 3 log CFU/ml, the biofilm levels of Salmonella on the stainless steel surface reached 4.5 log CFU/cm2 at 10 °C and 6.5 log CFU/cm2 at 22 °C in 7 days. No differences were observed between Salmonella strains tested for biofilm formation in catfish mucus extract on the stainless steel surface. The biofilm formation by Salmonella Blockley (7175) in catfish mucus extract was less (P < 0.05) on buna-N rubber when compared to stainless steel, polyethylene and polyurethane surfaces. Salmonella biofilm cells were not detectable on the stainless steel surface after treatment with a mixture of disinfectants but were still present when single compound disinfectants were used.