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

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 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 such as 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 in vitro 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 v/v 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. were 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 was 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 was also 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 μl of 10 μM forward primer, 1μl of 10 μM reverse primer and 8.5 μl of nuclease-free watering 2 μl 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 2 μl 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 at 4ºC. The results of the PCR reactions 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. Extracted DNA was sent to an external laboratory (Wright Labs or Novogene) for PCR amplification and sequencing of the 16S rDNA V4 region of the bacteria present in each sample.  

1.3 Bioinformatics. Sequences were analyzed with DADA2 pipeline following the standard protocol for 16S rRNA V4 region amplicon sequence reads. Paired-end sequencing reads were merged into contigs and contigs shorter or longer than 254 bp were discarded. Chimera were detected and removed. Amplicon sequence variants (ASVs) were assigned taxonomy by aligned against the reference database Silva (version 132). 

1.4 Statistical analyses of microbiome data. To avoid eliminating a subset of data through normalization or rarefaction, we used the microbiome compositional data analysis approach. All ASVs 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 data, Principal 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 R package compositions (version 1.40-5). The composition of microbiota for each facility was visualized at the family taxonomic level using stacked bar plots. Bacterial ASVs with less than 2% relative abundance were merged into the category “Others” and bar plots 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. Lactic acid bacteria (LAB) strains Lactococcus lactis ssp. lactis C-1-92 (LL) and Enterococcus durans 152 (ED) were obtained from the American Type Culture Collection (ATCC). 

2.2 Colonization of protective cultures in reconstituted dairy facility microbiome. To determine whether the two LAB strains can attach to a model surface (i.e., polypropylene) in the presence of microbiota collected from ice cream processing facilities, we added them to aliquots of composite microbiota suspensions. The two LAB cultures were added to microbiota each individually, as well as together. For each facility, the microbiota suspension prepared in 1.1 was aliquoted into 15-ml polypropylene conical tubes in duplicates. Tubes were inoculated with each LAB strain at a final concentration of 5*10⁷ CFU/ml to reach a total volume of 2 ml. Negative controls were not inoculated with the LAB strains to measure the effect of LAB strains on the composition of attached microbiota. Inoculated tubes were incubated at 15°C for 3 days to allow for attachment cells to the surface. The temperature of incubation was defined as the mean temperature measured in dairy processing facilities during sampling at the time of sample collection. After incubation, the medium was removed from tubes, and the attached biomass was washed twice with 0.1% peptone water to remove planktonic and loosely attached cells. The attached microbiota was resuspended in 2 ml of 0.1% peptone water by adding 2 g of 3 mm sterile glass beads and vortexing for 30 seconds. Ten-fold serial dilutions of resuspended microbiota were prepared in 0.1 % peptone water and spread plated in triplicate onto (BHI) agar to quantify the total amount of culturable bacteria and De Man, Rogosa, and Sharpe (MRS) agar to quantify LAB. The inoculated plates were incubated at 35°C for 3 days. Each experiment had two technical replicates per treatment and was conducted in two independent replicates. One milliliter of the resuspended attached microbiota suspension was used to characterize the microbial composition of attached biomass. DNA from the resuspended biomass was extracted using the DNeasy Power Biofilm kit (Qiagen, Germantown, MD). The extracted DNA was used for PCR-amplification of the 16S rRNA V4 region, library preparation, sequencing and analysis as described in 1.2 – 1.4. 

Objective 3: Measure the ability of protective cultures to inhibit L. monocytogenes in the presence of small-scale dairy environment microbiome biofilms. 

3.1 Listeria monocytogenes isolates. Eight Listeria monocytogenes strains of different linages that were previously isolated from dairy processing environments, were obtained from Dr. Martin Weidmann at Cornell University. 

3.2 Inhibition of L. monocytogenes by protective cultures in reconstituted dairy facility microbiome. To determine whether the two LAB strains can effectively inhibit L. monocytogenes when in the presence of the microbiota collected from ice cream processing facilities, we added them to aliquots of the microbiome suspensions, individually and combined. The microbiota suspension collected from each facility was prepared as outlined in 1.1 and was aliquoted into 15-ml conical polypropylene tubes in duplicate. Tubes were inoculated with each biocontrol strain at a final concentration of 5*10⁷ CFU/ml, and with of an 8-strain cocktail of L. monocytogenes at a final concentration of 3*10⁶ CFU/ml. Negative controls were not inoculated with the LAB strains or L. monocytogenes to measure the effect of the strains on the microbial composition of attached biomass. Positive controls were inoculated with L. monocytogenes without the addition of LAB strains. The tubes were incubated at 15°C for 3 days to allow for the attachment of cells to the surface. After incubation, the medium was removed, and the attached biomass was washed twice in 0.1% peptone water to remove planktonic and loosely attached cells. Attached cells were resuspended in 2 ml of 0.1% peptone water by adding 2 g of 3 mm sterile glass beads and vortexing for 30 seconds. Ten-fold serial dilutions of resuspended attached biomass were prepared in 0.1% peptone water and were spread plated in triplicate onto BHI agar. Inoculated plates were incubated at 35°C for 3 days. To quantify the concentration of L. monocytogenes in the attached biomass, a modified FDA BAM MPN protocol was used. For each treatment and the initial environmental microbiota suspension, 7-decimal dilutions were made in 0.1% peptone water. Then, 100 μl of each dilution were transferred to three microcentrifuge tubes containing 900 μl of Buffered Listeria Enrichment broth (BLEB), followed by incubation at 30°C for 4 hours. After incubation, 4 μl of Selective Listeria Enrichment Supplement (90 mg acriflavine, 450 mg cycloheximide and 360 mg sodium nalidixic acid in 40 ml of sterile DI water) was added to each tube followed by incubation at 30°C for 44 additional hours. After incubation, an aliquot of each tube was streaked onto Agar Listeria Ottavani & Agosti (ALOA) and incubated at 37°C for 48 hours. The presence of blue colonies with a halo indicated the presence of L. monocytogenes in the enrichment. The number of positive L. monocytogenes MPN tubes was recorded, and the final concentration was calculated using the MPN calculator available from the FDA BAM appendix 2: Most Probable Number from Serial Dilutions. The experiment was conducted in two independent biological replicates. One milliliter of the attached biomass suspension was used to characterize its bacterial composition. DNA from the attached biomass was extracted using the DNeasy Power Biofilm kit (Qiagen, Germantown, MD). Extracted DNA was used to PCR-amplification of the 16S rRNA V4 region, library preparation, sequencing and analysis as described in 1.2 – 1.4. 

Objective 4: Development of educational and outreach videos. 

Educational videos on the topics of "Listeria in the dairy industry" and "Biofilms in the dairy industry" were developed and will be made available to stakeholders via the Penn State Extension website. In addition, two extension articles were written to include with each video to provide additional information to dairy processors and additional resources. Both videos and extension articles were translated to Spanish.