Progress 07/01/21 to 06/30/23
Outputs
Target Audience: The target audiences that were the focus of effort for the duration of this projectincluded all stakeholders in the dairy food product industry including producers, regulators, retailers, industry groups, and educators. Producers were reached through extension, outreach, and consulting activities. Regulators, industry groups, scientists and educators were reached through presentations at an international food safety conference. Efforts on this project, including laboratory instruction, also reached undergraduate and graduate students involved in, and mentored during, this work. Changes/Problems:There were no major changes as described but minor changes are discussed here for clarity. We originally proposed to screen strains for phage sensitivity using a single agar overlay method. We felt that this was insufficient and decided to add another screening method based on broth microdilution to better asses the ratio of phage to pathogen needed for subsequent experiments in milk. Results from these screening experiments suggested that most strains were similarly lysed by the phage products such that we were unable to clearly characterize five as most resistant as proposed. Instead, subsequent trials in milk included three to four strains each to represent low, medium and high relative phage sensitivity to better evaluate and compare the impact of growth matrix (broth vs. pasteurized milk vs. raw milk). Preliminary data also resulted in the selection of a single inoculation level for milk experiments (2 log CFU/mL) as we were unable to effectively enumerate survivors in trials inoculated at 1 CFU/mL.We also selected a single MOI based on the results from the additional screening method employed and preliminary trials that showed phage addition at <8 log PFU/mL was ineffective. Preliminary experiments using a cheesemaking temperature profile for incubation also indicated that the phage activity occurred during the first 4 h and were not affected by temperature. This proposed approach also failed to consider the impact of coagulation during cheesemaking on the phage-pathogen interactions. Data from the milk storage experiments conducted at 7°C were deemed sufficient for our project goals and more applicable considering experiments in cheese were to be conducted. Because only theSalmonellaphage product was effective in raw milk, we only conducted raw milk cheese trials withSalmonella. This potential shortcoming was expected and included in the proposal. Serovar Montevideo was selected for these trials based on data generated in milk but this strain was unable to grow in raw milk Queso Fresco and declined rapidly in raw milk Gouda. This was also anticipated and still demonstratedan inability of phage addition to further reduce pathogen counts in cheese, which remains an impactful result. What opportunities for training and professional development has the project provided?The project provided one on one training opportunities for one graduate student as well as two undergraduate students increasing their proficiency in agriculture related laboratory research. The graduate students working on this project attended the 2022 International Association for Food Protection Annual Meeting to present their results. This provided a unique opportunity for professional development to increase their knowledge and expertise in the field of food safety and microbiology. How have the results been disseminated to communities of interest?Results and other insight gained from this research have been shared with stakeholders through the PIs extension work with small-scale value-added dairy processors. We were able to reach a number of stakeholders thorough educational presentations at the 2022 International Association for Food Protection Annual Meeting. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Knowledge was gained on the antimicrobial activity and host range of commercial bacteriophage products againstListeria monocytogenes, Shiga toxin-producingEscherichia coli(STEC) and serovars ofSalmonellaentericarelevant to the dairy industry. We also generated new knowledge regarding the role of phage screening methods as they relate to the results observed in food matrices. It is expected that this change in knowledge will lead to a change in action once shared. Graduate and undergraduate students also gained knowledge on microbiological techniques and research methods. Determine the antimicrobial activity of commercial phage preparations againstListeria monocytogenes,SalmonellaandSTECin raw milk. With the assistance of graduate and undergraduate research students, we determined the titers of three commercially available phage products (one each forSalmonella, STEC, andL. monocytogenes) and the resulting efficiencies of plating (ratio of titer to a reference strain) for 10 strains (9 serovars) ofSalmonellaenterica, 18 strains ofL. monocytogenes, 11 strains ofE. coliO157:H7, and 6 strains of other STEC serotypes (O103, O45, O145, O26, O111, and O121). Results identified differences in the EOP between strains of each pathogen. For example the EOPs ranged from 0.92-0.99, 0.87-0.99, and 0-0.98 forL. monocytogenes,E. coliO157:H7 andSalmonella, respectively. Only one strain ofSalmonella(serovar Cerro) did not produce titers, whereas titers were not identified using STEC O111, O26, or O121. The EOP for the remaining STEC ranged from 0.94-0.99, demonstrating a larger range of susceptibility among STEC.These results suggest that the host range for this phage product is relatively broad among the strains tested with few exceptions, notably among non-O157 STEC. We further determined the phage counts needed to lyse each strain in laboratory media from a starting inoculation of 2 log CFU/mL using a broth microdilution assay with visual turbidity as the endpoint. The ratio of the phage (PFU/mL) needed for lysis to the starting bacterial inoculation (CFU/mL) was considered as the effective MOI expressed as log PFU/CFU. The MOI forL. monocytogenesphage product ranged from 2.53 log to 3.59 log PFU/CFU with one strain (serotype 7) that was not inhibited at the maximum phage application (8 log PFU/mL, MOI 6 log PFU/CFU). Larger ranges in MOI were identified for bothSalmonellaandE. coliO157:H7 (0.99-4.57 and 1.0-5.12 log PFU/CFU, respectively).MOI could only be determined for the same three serotypes of non-O157 STEC (O103, O45, and O103) from which titers were determined and ranged from 2.47-4.21 log PFU/CFU. Overall, titer/EOP results did not always align with the MOI results. Together, these data suggest that the use of one screening tool is insufficient and that each approach may yield differing results. Strains were then selected to represent those with relatively high to low resistance for determining the antimicrobial efficacy in both raw and pasteurized milk. The respective phage products were added to pasteurized milk containing 2 log CFU/mL of eitherSalmonella, STEC O157:H7, orL. monocytogenes(MOI of 6 log PFU/CFU) and held at 7? for 1 week. Phage counts remained near inoculation levels throughout storage. Mean counts of three different strains ofL. monocytogenesin pasteurized milk decreased 0.6-0.7 log CFU/mL from inoculation to the 1 h timepoint and differed significantly from control at 4 h (~1.5 log CFU/mL reductions from inoculation). Mean counts in treatments did not change through the remainder of storage whereas counts in the controls increased ~ 2 log CFU/mL. Counts of two of three O157 strains in pasteurized milk without phage did not change during storageat 7°C for 7d, whereas counts of the third strain were ~0.5 log CFU/mL lower than inoculation. The addition of phage significantly reduced counts at hours 1, 24, or 72, depending on the strain, when compared to control.There was no regrowth for the three treated strains by the end of the study resulting in counts 0.5-2 log CFU/mL lower than control at the end of the study. TheSalmonellabacteriophage preparation was more effective with significant reductions in counts at 1 h for serovars Newport, Montevideo, and Typhimurium. However, counts of serovar Cerro were not significantly lower than inoculation until 4 h. Counts of all serovars in phage treatments differed from control at all time points after 4 h and remained similar over time, whereas counts in controls increased steadily over 7 days. The same approach was applied in raw milk. In contrast to pasteurized milk,E. coliphage counts decreased ~1.5-2 log PFU/mL by 24h and remained at that level throughout storage whereas theL. monocytogenesandSalmonellaphages decreased the same amount but gradually over the 7 d. Pathogen counts in controls did not change or decreased steadily over time. Despite the efficacy in pasteurized milk, there was no effect of phage application on counts ofL. monocytogenesor STEC O157:H7 in raw milk. However, addition of theSalmonellaphage product resulted in reductions in serovars Newport, Montevideo, and Typhimurium to counts below the limit of enumeration at 1 h and remained unchanged throughout the remainder of the study. In contrast, no differences in counts between treatment and control were observed for serovar Cerro. Results for the strains tested in milk were relatively consistent with MOI data, whereby strains ofL. monocytogeneswere affected relatively equally. Similarly,SalmonellaTyphimurium was most sensitive to the phage, andSalmonellaCerro was most resistant. 2.Determine the antimicrobial activity of commercial phage preparations againstListeria monocytogenes,SalmonellaandSTECduring the manufacture and storage of raw milk cheese. Based on the results from experiments in raw milk, only theSalmonellaphage was tested in the production of raw milk Queso Fresco (pH ~6.4, 49% moisture) and Gouda (pH ~5.1, 44% moisture) cheese. Raw milk was inoculated with 1-2 log CFU/mL ofSalmonellaMontevideo and 8 PFU/mL of the phage preparation. Despite the observations in raw milk,Salmonellacounts in control and phage treated Queso Fresco remained relatively unchanged from the initial milk inoculation of 2.02-2.08 log CFU/mL to 2.42-2.84 log CFU/g remaining in the cheese on day 1. Phage counts decreased significantly between inoculation (8.23 log PFU/mL) and coagulation (6.75 log PFU/mL) with similar titers in the finished cheese (5.8 log PFU/mL). Phage counts held at ~6 log PFU/mL during 28 days of storage at 7°C whereas Salmonella counts in the control decreased 1.6 log CFU/g during. Counts also decreased 1.67 log CFU/g in the phage treated cheese. The decrease in counts was similar but final counts were lower in phage treated cheese as they started lower. In the second set of experiments, raw milk for the production of Gouda cheese was inoculated withSalmonellaMontevideo at either 1 (low) or 2 (high) log CFU/mL. Changes in phage titers were similar in the production of Gouda; decreasing significantly from inoculation to coagulation and ending at ~6 log PFU/mL in the cheese on day 1.Salmonellacounts fell below the limit of enumeration before brining in phage treated cheese and after brining (day 1) in the control when raw milk was inoculated at ~1 log CFU/mL. Results for the higher inoculation level were similar exceptSalmonellawas present at ~1 log CFU/g in the control cheese after brining on day 1 but below the limit of enumeration at day 7.Salmonellain cheese at the low inoculation level were detectable by enrichment longer in phage treated cheese (day 60) compared to control (day 28).Despite the slightly higher counts on day 1,Salmonellain the high inoculation trials were detectable by enrichment in both cheeses up to day 60. Publications resulting from this work are in preparation for submission.
Publications
|
Progress 07/01/21 to 06/30/22
Outputs
Target Audience:The target audience reached during this reporting periodincludedall stakeholders in the dairy food product industryincluding producers, regulators, retailers, industry groups,and educators. Producers were reached through extension, outreach, and consulting activities. Regulators, industry groups,scientists and educators were reached through presentations at an international food safety conference. Efforts on thisproject, including laboratory instruction, also reached undergraduate and graduate students involved in, and mentoredduring, this work. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided one on one training opportunities for one graduate students as well as one undergraduate student,increasing their proficiency in agriculture related laboratory research. The graduate studentworking on this project were also able to attend the 2022International Association for Food Protection Annual Meeting to present their results. This meeting alsoprovided a unique opportunity for professional development to increase their knowledge and expertise in the field of food safetyand microbiology. How have the results been disseminated to communities of interest?Results and other insight gained from this research have been shared with stakeholders through the PIs extension work withvalue-added dairy processors. We were able to reach a varietyof stakeholders thoroughpresentations at the 2022 International Association for Food Protection Annual Meeting. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will complete the screening trials and select pathogen strains and their respective bacteriophage preparations to be used in cheese manufacturing experiments. We will then determine the effect of bacteriophage preparations against L. monocytogenes, Salmonella, and/or STEC during the manufacture and storage of cheese manufactured from raw milk.
Impacts
What was accomplished under these goals?
Knowledge was gained on the antimicrobial activity and host range of commercial bacteriophage products against Listeria monocytogenes, Shiga toxin-producing Escherichia coli (STEC) and serovars of Salmonella enterica relevant to the dairy industry. WE also generated new knowledge regarding the role of phage screening methods as they relate to the effects observed in food matrices. It is expected that this change in knowledge will lead to a change in action once shared. Graduate and undergraduate students also gained knowledge on microbiological techniques and research methods. Determine the antimicrobial activity of commercial phage preparations againstListeria monocytogenes, SalmonellaandSTECin raw milk. With the assistance of graduate and undergraduate research students, we determined the titers of three commercially available phage products (one each for Salmonella, E. coli, and L. monocytogenes) and the resulting efficiencies of plating (ratio of titer to a reference strain) for 10 strains (9 serovars) of S. enterica, 18 strains of L. monocytogenes, 11 strains of E. coli O157:H7, and 6 strains of other STEC serotypes (O103, O45, O145, O26, O111, and O121). Results identified some differences in the EOP between strains of each pathogen. For example the EOPs ranged from 0.92-0.99, 0.87-0.99, and 0-0.98 for L. monocytogenes, E. coli O157:H7 and Salmonella, respectively. Only one strain of Salmonella (serovar Cerro) did not produce titers, whereas titers were not identified using STEC O111, O26, or O121. The remaining EOP for STEC ranged from 0.94-0.99, demonstrating a larger range of susceptibility among STEC compared to O157:H7.These results suggest that the host range for this phage product are relatively broad among the strains tested with few exceptions, notably among non-O157 STEC. We further determined the phage counts needed to inhibit the growth of each strain in laboratory media from a starting inoculation of 2 log CFU/mL using a broth microdilution assay. The ratio of the inhibitory level of phage (PFU/mL) to the starting bacterial inoculation (CFU/mL) was considered as the effective MOI expressed as log PFU/CFU. The MOI for L. monocytogenes phage ranged from 2.53 log to 3.59 log PFU/CFU with one strain (SLCC) that was not inhibited at the maximum phage application (8 log PFU/mL, MOI 6 log PFU/CFU). Larger ranges in MOI was identified for both Salmonella and E. coli O157:H7 (0.99-4.57 and -0.51-5.07 log PFU/CFU, respectively).MOI could only be determined for the same three serotypes of non-O157 STEC (O103, O45, and O103) ranging from 1.66-4.21 log PFU/CFU. Overall, titer/EOP results did not always align with the MOI results. For example, the L. monocytogenes strain with the lowest EOP (493), which suggests it is relatively resistant, also had one of the lowest MOIs, which suggests it is one of the most sensitive. Similarly, we could not identify an MOI for strain SLCC despite having an EOP of 0.94. An MOI of 5.67 log PFU/CFU was identified for S. enterica serovar Cerro despite the inability to enumerate titers with this strain. Together, these data suggest that determination of MOI using the method applied is better suited for differentiating host susceptibility and as a screening tool for subsequent applications. Strains were then selected to represent those with high to low resistance for determining the antimicrobial efficacy in both raw and pasteurized milk. The first set of trials were conducted in pasteurized milk as the literature suggests phage activity may be attenuated in raw milk. The respective phage products were added to pasteurized milk containing 2 log CFU/mL of either S. enterica or L. monocytogenes (MOI of 6 log PFU/CFU) and held at 7?. Mean counts of three different strains of L. monocytogenes in pasteurized milk decreased 0.6-0.7 log CFU/mL from inoculation to the 1 h timepoint and differed significantly from control at 4 h (~1.5 log CFU/mL reductions from inoculation). Mean counts in treatments did not change through the remainder of storage whereas counts in the controls increased ~ 2 log CFU/mL. The Salmonella bacteriophage preparation was more effective with significant reductions in counts at 1h for serovars Newport, Montevideo, and Typhimurium. However, counts of serovar Cerro were not significantly reduced until 4 h. Counts of all serovars in phage treatments differed from control at all time points at 4 h and remained similar over time, whereas counts in controls increased steadily over 7 days. The same approach was applied in raw milk. In contrast to pasteurized milk, pathogen counts in controls did not change or decreased steadily over 5 days. Despite the efficacy in pasteurized milk, there was no effect of phage application counts of L. monocytogenes in raw milk. However, addition of the Salmonella phage product resulted in reductions in serovars Newport, Montevideo, and Typhimurium to counts below the limit of enumeration at 1h and remained unchanged throughout the remainder of the study. In contrast, no differences in counts between treatment and control were observed for serovar Cerro. Results for the strains tested in milk were relatively consistent with MOI data, whereby strains of L. monocytogenes were affected relatively equally. Similarly, S. Typhimurium was most sensitive to the phage, and S. Cerro was most resistant. Overall, the work conducted thus far demonstrates the host range of three commercially available phage products for the control of their respective bacterial pathogens in an intended dairy matrix. Differences in effectiveness against different strains/serovars and in different matrixes (pasteurized vs. raw milk) inform future applications in the production of cheese and other dairy products. 2.Determine the antimicrobial activity of commercial phage preparations againstListeria monocytogenes, SalmonellaandSTECduring the manufacture and storage of raw milk cheese. Nothing to report during this reporting period for this goal.
Publications
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2022
Citation:
Everhart, E., S. Carson, and D. DAmico. 2022. Commercial Bacteriophage Preparations for the Control of Listeria monocytogenes in Raw and Pasteurized Milk. International Association for Food Protection Annual Conference 2022.
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2022
Citation:
Worth, A., E. Everhart, and D. DAmico. 2022. Effect of a Commercial Bacteriophage Preparation Against Dairy-Relevant Salmonella Enterica Serovars in Raw and Pasteurized Milk.International Association for Food Protection Annual Conference 2022.
|