Using Protective Cultures to Control Listeria monocytogenes in Microbiomes from Small-Scale Dairy Production Facilities

Progress report for GNE19-215

Project Type: Graduate Student
Funds awarded in 2019: $14,940.00
Projected End Date: 07/31/2022
Grant Recipient: The Pennsylvania State University
Region: Northeast
State: Pennsylvania
Graduate Student:
Faculty Advisor:
Jasna Kovac
The Pennsylvania State University
Expand All

Project Information

Project Objectives:

Objective 1: Characterization of small-scale dairy processing facility microbiomes.

-Expected outcome: Characterization of environmental microbiomes collected from small-scale dairy processing environments.

-Deliverables: Microbiome sequences made publicly available; table with relative abundances of bacteria and results of statistical analyses of microbial diversity.

-Potential pitfalls and alternative approaches: I have learned microbiome characterization methods while assisting with a project in apple-packing facilities. Dairy processing environments will likely contain less soil, but more fat compared to apple facility samples, which may act as PCR inhibitors. If this occurs, I will further optimize PCR conditions and test different master mixes that are less susceptible to the inhibitors.

Objective 2: Measure the ability of protective cultures to effectively colonize small-scale dairy environment microbiome biofilms.

-Expected outcome: Protective cultures will colonize the dairy processing environment microbiome biofilms and remain in high concentration in a microbiome biofilm within a week period.

-Deliverable: Spreadsheet with quantification results of protective cultures in biofilms of small-scale dairy facility microbiomes.

-Potential pitfalls and alternative approaches: If the cultures selected do not show the ability to effectively colonize the environmental biofilm, other lactic acid bacteria will be tested. We will also attempt to isolate other bacteria from small-scale environment microbiomes, since native strains will more likely be able to survive and remain active in similar environments.

Objective 3: Test the efficacy of protective cultures against Listeria monocytogenes (Lm) within an in vitro grown dairy environment microbiome biofilm.

-Expected outcome: Protective cultures will be able to inhibit the survival and growth of Lm in dairy processing facility microbiome biofilms.

-Deliverables: Spreadsheet with log10MPN reduction of Lm in facility microbiome biofilms after adding protective cultures.

-Potential pitfalls and alternative approaches: If the selected tested cultures do not show the ability to effectively colonize the environmental biofilm, we will test other lactic acid bacteria, including those isolated from sampled small-scale dairy processing environments.

Objective 4: Development of educational materials for dissemination of results among stakeholders.

-Expected outcomes: Educational video covering cleaning and sanitation practices for control of Lm and fact sheets will be developed and disseminated among stakeholders through Penn State Extension workshops.

-Deliverables: Educational video, fact sheets and workshop lectures.

-Potential pitfalls and alternative approaches: If the proposed biocontrol strategy does not show promising results, we will still share information on good hygiene and sanitation practices with shareholders, as this can help them improve safety of their food products.

Introduction:

Pennsylvania produces over 15% of the total milk supply in the U.S. and 99% of Pennsylvania dairy farms are family owned. Many of them are small farms that process the milk they produce to add value to their product. However, previous extension research from Penn State indicates that small-scale dairy processors face food safety challenges that can compromise their businesses. One of the main microbiological food safety hazards associated with the dairy supply chain is Listeria monocytogenes (Lm). The Center for Disease Control and Prevention estimates that out of 1,591 cases of human listeriosis occurring every year in the United States, 260 result in death. Lm has been associated with dairy foods, including soft cheeses, ice cream and raw milk. The presence of Lm in ready-to-eat food may result in recalls that generate an economic loss to producers, as well as distrust of their brand’s image. It has been estimated that the annual cost of listeriosis in the United States 2.6 billion dollars. 

While Lm is effectively inactivated during pasteurization, it may be re-introduced to food products when present in the food processing environment. It is therefore critical for food processors to effectively control Lm presence in the environment to prevent contamination of products and maintain consumer trust. However, it is particularly challenging to eradicate Lm from food processing environments, since it can survive and grow in cold and humid environments, such as dairy processing facilities. While Lm is susceptible to common cleaning and sanitizing practices applied by the dairy industry, it can grow in biofilms with bacterial communities present in the processing environments. Biofilms are attached aggregates of bacteria that secrete slime-like extracellular polymeric substances that bind them together and protect them from environmental stressors such as cleaners and sanitizers. Lm can benefit from the interactions with certain environmental microbiomes, such as Pseudomonas, since Pseudomonas is a very good biofilm former that can provide protection to Lm. Nevertheless, these interactions with environmental microbiome can also be leveraged in food processors’ advantage, if protective cultures that can outcompete or inhibit the growth of biofilm formers and Lm are added to the environment. It is known that certain microbial species, including lactic acid bacteria, can suppress the growth and adhesion of Lm. I therefore propose to test two biocontrol strains that had previously been shown to be effective in inhibition of Lm in poultry processing facilities. If successful, implementation of such protective cultures in the food processing environment may provide enhanced benefits compared to sanitizers alone, since biocontrol strains have the added advantage of not promoting antimicrobial tolerance and resistance development in Lm and will likely be effective also against antimicrobial-resistant Lm strains. If the results show merit in our in vitro study, these interventions may be tested in the future in actual facility environments. his approach is considered sustainable, because it is based on natural interactions among beneficial microorganisms that can be leveraged in small-scale dairy processors’ advantage. Specifically, application of protective biocontrol cultures is less likely to facilitate the development of Lm resistance, which often occurs as a result of chemical treatments.   

While I am not seeking to replace traditional sanitation methods, I believe biological control can be use in addition to physical and chemical interventions to enhance their efficacy. This project will be the first to test the efficacy of protective cultures in conjunction with dairy facility microbiome. If successful, I will continue to investigate the application of biocontrol agents within an actual dairy processing facility. In addition to research, I intend to develop educational materials focused on environmental control of Lm. These will communicate the importance of cleaning and sanitation practices to reduce Lm incidence in food processing environments, as well as explain how microbiome biofilms can contribute to persistence of Lm in food processing facilities. It also will be important to inform the end users in how biological control interventions should be without compromising the facility hygienic status.  Overall, I believe this project is an important first step towards providing small-scale processors with new strategies for Lm control, resulting in enhanced sustainability of their business through improved food safety. 

Cooperators

Click linked name(s) to expand
  • Dr. Robert Roberts (Educator and Researcher)
  • Dr. Kerry Kaylegian (Educator and Researcher)

Research

Materials and methods:

Description of materials and methods used for this project in the period August 2019 to January 2021. The remaining methods will be added in the next annual report to show the progress of the project.

Objective 1: Characterization of small-scale dairy processing facility microbiome. 

 1.1 Sample collection.  Three small-scale ice cream processing facilities were enrolled in this study. These facilities are small to medium-size operations, that produce a variety of dairy products, including milk, cheese and, ice cream. Six samples for microbiome analyses were collected from each facility using 3M hydrated sponges with neutralizing buffer by a combination of ten horizontal and 10 vertical swabbing moves form an area of 40 cm by 40 cm or an equivalent area in sites that are not flat. Sampled areas included non-food contact surfaces including drains and floors among others, within the ice cream processing room in each facility. Samples were stored on ice in a cooler during the transportation to the lab. During sampling, the temperature of each dairy processing facility was measured to adjust the incubation temperature of our biofilm experiments (Obj. 2 and 3) to represent dairy processing facility conditions. Ninety milliliters of Brain Heart Infusion (BHI) broth were added to each of the six samples collected per facility. Samples were homogenized using a laboratory stomacher for 14 minutes at 230rpm, to assure that all soil was transferred to the media, and homogenates of all samples were combined to obtain a microbiome sample representative of each facility. Twenty percent glycerol was added to homogenates to allow for preservation at -80 °C. 

1.2 DNA extraction and 16S rDNA extraction for microbiome sequencing. Fifty milliliters of the homogenate prepared in 1.1. was transferred to a sterile 50 ml conical tube and centrifuged at 11,000 g and 4 ºC for 20 minutes. After centrifugation, the supernatants were discarded and pellets containing microbiomes were stored at -80 °C until DNA extraction. DNA will be extracted from approximately 0.25 g sample using Omega E.Z.N.A Soil DNA kit. A sterile sponge (representative of a production lot) homogenized in BHI will also be processed, as a negative control, to confirm absence of microbial DNA contaminants in the sterile sponge and BHI. Concentration of DNA after extraction was determined with Nanodrop One and using Qubit dsDNA High Sensitivity Assay Kit. PCR was used to amplify the16S rDNA V4 region of the bacteria present in each sample. Briefly, the V4 region of the 16S rRNA gene sequence was amplified using a forward primer 505F-v2 (TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG GTG YCA GCM GCC GCG GTA A) and a reverse primer 806R-v2 (GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGG ACT ACN VGG GTW TCT AAT). The PCR mastermix was composed of 12.5ul of KAPA 2x KAPA HIFI HotStart Ready Mix , 1 ul of 10uM forward primer, 1ul of 10uM reverse primer and 8.5ul of nuclease-free watering 2ul of the extracted DNA were added per reaction. A positive control (microbiome sample from an apple packing house that was obtained following the same sampling protocol) was included in each run, as well as a negative control with 2ul of nuclease free water. PCR thermal cycling for amplification of the 16S rRNA gene V4 region was conducted as follows: initial denaturation at 98ºC for 2 min, 30 cycles of denaturation at 98ºC for 20 s, annealing at 60ºC for 20 s, extension at 72ºC for 25 s, and final extension at 72ºC for 5 min; final hold was atC. The results of the PCR reaction were visualized on a 2% agarose gel electrophoresis run at 130V for 30 minutes to confirm successful amplification. Concentration of DNA amplicons will be determined by Qubit 3 using Qubit dsDNA High Sensitivity Assay Kit. 

1.3 Bioinformatics. Sequences were be analyzed with Mothur v1.42.3 pipeline following the standard protocol for 16S rRNA V4 region amplicon sequence reads. Pairedend sequencing reads were merged into contigs and contigs shorter or longer than 292 bp were discarded. The sequences remaining after filtering were aligned against the reference database Silva (version 132)prior to OTU picking. Chimera were detected and removed using the VSEARCH algorithm. Bacterial OTUs were clustered using the opticlust algorithm with 97% similarity threshold and clustered OTUs were assigned taxonomy. 

1.4 Statistical analyses of microbiome data. Alpha diversity was calculated using the Chao1 index to estimate the species richness and compared to the number of observed OTUs in each sample to calculate the percentage of diversity that was discovered by the sequencing effort. To avoid eliminating a subset of data through normalization or rarefaction, we used the microbiome compositional data analysis approachAll OTUs with “0” count value were assigned a small, non-zero value using the Count Zero Multiplicative Method with the zCompositions package (version 1.3.4) prior to applying the center-log ratio (clr) transformation. To visualize the dataPrincipal Component Analysis (PCA) was used on clr-transformed data.To explore the taxonomic compositions of samples, relative abundances of taxa were calculated using the Aitchison Simplex method with the package compositions (version 1.40-5)The composition of microbiota for each facility was visualized at the family taxonomic level using stacked barplots. For the visualization purposes, the OTU tables were collapsed to the family level with the phyloseq package (version 1.28.0). Bacterial families or genera with less than 1% relative abundance were merged into the category “Others” and barplots were generated with the package ggplot2 (version 3.3.2) 

Objective 2: Measure the ability of protective cultures to effectively colonize small-scale dairy environment microbiome biofilms. 

 2.1 Putative protective cultures and Listeria monocytogenes isolates. Lactococcus lactis ssp. lactis C-1-92 and Enterococcus durans 152 were obtained from the ATCC bacterial culture collection. Eight Listeria monocytogenes cultures of two different linages that were previously isolated from dairy processing environments, were obtained from Dr. Martin Weidmann at Cornell University. 

2.2 Colonization of protective cultures in reconstituted dairy facility microbiome. To determine whether the two putative protective cultures can colonize biofilms with the microbiomes of ice cream processing facilities, we added them to aliquotes of the microbiome suspensions individually and combined. For each facility, 2 ml of the microbiome suspension prepared in 1.1 was aliquoted into 15-ml conical tubes in duplicate. Tubes were inoculated with 10^5 CFU/ml of each biocontrol strain, or with an equal ratio mixture of both strains at a final concentration of 10^5 CFU/ml. Negative controls were not inoculated with the putative biocontrol strains to allow for comparison of the effect of the strains on the biofilm composition. The tubes were incubated at 15°C for 3 days to allow for biofilm development. The temperature of incubation was defined as the mean temperature measured in the dairy processing facilities during sampling visits. After incubation, the medium was removed and the biofilms were washed twice in 0.1% peptone water to remove planktonic and loosely attached cells. Biofilms were resuspended in 2 ml of 0.1% peptone water by adding 2 mm sterile glass beads and vortexing for 30 seconds. Serial dilutions of resuspended biofilm biomass were preapred in 0.1% peptone water and be spread plated in triplicate onto Brain Heart Infusion (BHI) agar to quantify the total amount of culturable bacteria and De Man, Rogosa and Sharpe (MRS) agar to quantify lactic acid bacteria (a group of bacteria that includes both protective cultures). Plates were incubated at 35°C for 2 days. The experiment was conducted in two independent biological replicates. One mililiter of the biofilm suspension was used to charactize the composition of the biofilms. DNA from the bioiflms was extracted using the DNeasy Power Biofilm kit (Qiagen, Germantown, MD) followed by amplification of the 16S rRNA V4 region, library preparation, sequencing and analysis as described in  1.2 – 1.4. 

Research results and discussion:
 

Objective 1: Characterization of small-scale dairy processing facility microbiome. 

Three small scale ice cream processing facilities were enrolled to participate in this study (the facilities are coded as “A”, “B”, and “C” thorughout the report to preserve confidenciality). Six samples were collected from non-food contact surfaces including floors and drains within the ice cream processing environment of each facility and combined into one composite sample for microbiome characterization. Alpha diversity of each sample was calculated using the Chao1 index, which estimates the total number of species expected in each sample. The Chao1 index for the samples was 1336.9, 1010.0 and 1059.7 for facility A, B and C, respectively. Sequencing of the samples allowed to discover 61.6, 72.0 and 61.3 % of the OTUs present in Facility A, B, and C, respectively. 

To characterize and compare the composition of the microbiota between ice cream processing facilities, we analyzed the sequencing data using the compositional data analysis approach. Center-log ratio transformation was performed to the OTU tables. To visualize the taxonomic composition of the microbiota of the ice cream processing environments, we examined the relative abundance at the family level. In Facility A, 12 bacterial families were present at a relative abundance above 1%. Pseudomonadaceae was the predominant family in this facility (31.8% relative abundace), followed by Alternomonadaceae (14.6%) and Aeronomandaceae (11.9%) (Fig. 2). In facility B, 14 bacterial families were present at a relative abundance above 1%. Pseudomonadaceae was the predominant family in this facility (60.6%), followed by Enterobacteriaceae (5.6%) and Burkholderiaceae (5.1%) (Fig. 2). In Facility C, 16 bacterial families were present at a relative abundance above 1%. Rhodobacteriaceae was the predominant family in this  facility (27.7%), followed by Micrococcales_unclassified (12.5%) and Micrococcaceae (11.6%) (Fig. 2).

 

 

Fig. 2. Microbiome composition of ice cream processing facilities. Stacked bar plot showing relative abundance of bacterial families in a composite sample collected from three ice cream processing facilities (A, B, C). All families with less than 1% relative abundance were merged into “Others”.

Objective 2: Measure the ability of protective cultures to effectively colonize small-scale dairy environment microbiome biofilms.

The putative protective cultures Lactococcus lactis subsp. lactis and Enterococcus durans, that have been shown to succesfully control Listeria monocytogenes in drains from a poultry processing facility, were obtained from ATCC. To measure whether these putative protective cultures can colonize biofilms formed by the microbiomes of small-scale ice cream processing facilities, we cultured them independently (i.e., added one strain to the microbiome) and together. We first quantified the amount of culturable organims present in the composite microbiome samples of each facility (Fig.3). Total aerobic bacteria counts represent all the bacteria that can grow aerobically at the temperature of incubation, 15°C, while lactic acid bacteria count represent the bacteria that can grow on MRS medium (which is a selective medium for lactic acid bacteria). Facilities A and C were not significantly in their aerobic plate count (Fac. A = 6.97 logCFU/ml; Fac. C=6.61 logCFU/ml). Facility B had a significantly lower aerobic plate count than the other two facilities in this study (Fac. B=5.14 logCFU/ml). However, all facilities were not significantly different in their lactic acid bacteria count (Fac. A = 4.92 logCFU/ml; Fac. B = 4.58 logCFU/ml; Fac. C=4.61 logCFU/ml).

 
Fig. 3. Plate counts of microbiome composite samples from ice cream processing facilities. Aerobic plate counts and lactic acid bacteria counts of composite samples obtained from the environments of three ice cream processing facilities (A, B and C).

 

Microbiome composite samples were allowed to form biofilms in the presence of the putative protective cultures for 3 days at 15°C. A negative control was included to determine whether there was an increase in the total aerobic bacteria count and lactic acid bacteria count when in the presence of the protective cultures. For all facilities, there was a significant increase (p<0.05) in the total aerobic bacteria count and lactic acid bacteria count for the biofilms that were in the presence of putative protective cultures (Fig. 4). However, there was no significant difference between treatments, indicating that putative protective cultures can attach to biofilms of the environmental microbiomes of ice cream processing facilities either alone, or when combined.  

 

 

Fig. 4. Plate counts of biofilms formed by microbiomes collected from ice cream processing facilities in the presence of protective cultures. Aerobic plate counts and lactic acid bacteria counts of biofilms formed by the microbiomes collected from ice cream processing facilities in the presence of the protective cultures E. durans and L. lactis. NC: negative control (microbiome with no protective cultures added); LL: microbiomes with the addition of L. lactis; ED: microbiomes with the addition of E. durans; ; LE: microbiomes with the addition of L. lactis and E. durans.

To further visualize the composition of the biofilms, we extracted DNA and sent the samples for sequencing. Over the next year, I will recieve and analyze these results, which will further inform our discussion of the results.

 

Objective 3: Test the efficacy of protective cultures against Listeria monocytogenes (Lm) within an in vitro grown dairy environment microbiome biofilm.

I have obtained eight isolates of Listeria monocytogenes that have been previously isoated from dairy processing facilities by the group led by Dr. Martin Wieldamnn at Cornell University. These will be used over the coming year to test the effectiveness of these putative protective cultures within a biofilm of ice cream processing facility environmental microbiome. 

 

Objective 4: Development of educational materials for dissemination of results among stakeholders.

I am currently developing a script for an animated video on the topics of Listeria contamination in processing environments, roles of biofilms/microbiomes and, cleaning and sanitation methods used in the dairy industry including biocontrol methods. We expect that the video will be finalized by March 2021, and made available to dairy processors and the general public through the Penn State Extension website and the Kovac Lab YouTube channel. Further, these materials will be used when we resume the delivery of in-person workshops to dairy processors.

_______________

Given the University closure due to the COVID-19 pandemic, the work proposed for Obj 2 and 3 has been significantly delayed. 

Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary

Education/outreach description:

As outlined in my proposal, I am developing videos for Penn State Extension on the topics of Listeria contamination in processing environments, roles of biofilms/microbiomes and, cleaning and sanitation methods used in the dairy industry including biocontrol methods (Obj. 4). In addition, I will interview Penn States’ extension and education agents to learn about the most sucessfull strategies to comunicate this topics to farmers and processors. I will later apply these ideas in the development of the extension and outreach materials.

 

Project Outcomes

Project outcomes:

It is early to measure the project outcomes, as we have not gathered all the experimental data proposed.

Knowledge Gained:

Over the course of this project I have acquired skills in molecular biology and methods to sample and characterize environmental microbiomes, including DNA extraction methods, sequencing and bioinformatics. I have also taken courses in Agriculture Social Change applied to international development. The concepts learned in these course can be applied in this project.

During my doctoral degree, I plan to continue to investigate on the role of environmental microbiomes regarding food safety of food processing facilities. I am looking forward to applying novel genomic techniques to enhance food safety in dairy processing plants. Furthermore, I am interested in learning and applying advanced microbiological methods, including the use of molecular biology and environmental microbiology, to solve technical issues related tofood processing, quality and safety. I am particularly interested in generating tailored-solutions the development of sustainable food systems, and to mitigate food waste. I aim to become a scientific official for a development organization, such as the Food and Agricultural Organization, so that I may assist with developing sustainable food systems.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.