SOW_1229150_06_10_2020.pdf

Statement of Work_1229150

Food and Drug Administration (FDA) Center for Food Safety and Applied Nutrition (CFSAN)

Statement of Work

Characterization of the environmental microbiota composition and the presence of Listeria monocytogenes in commercial tree fruit packinghouses

I. Purpose

The purpose of this follow-on requirement is to continue to monitor total bacterial and fungal communities in three (3) commercial tree fruit packing facilities’ environments that might be associated with the persistence of L. monocytogenes by supporting the establishment and biofilm growth.

II. Background

Food and Drug Administration (FDA) and the Pennsylvania State University research teams have been working with three tree fruit packers in Pennsylvania for the past three years to establish a baseline prevalence of L. monocytogenes. While we found L. monocytogenes in all of them, some facilities had alarmingly high occurrence and concentrations of the pathogen in the packing house environment (~105 CFU/sponge swab), particularly directly underneath the roller brush conveyors that transport the apples through the washing, drying, and waxing process. Furthermore, our monitoring of total bacterial and fungal communities in these facilities’ environments indicated that environments with high occurrence of L. monocytogenes also have distinct microbiomes, which possibly support biofilm growth due to high relative abundance of Pseudomonadaceae (https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-019-0726-2). In cases when microbiota in the environment forms biofilms, L. monocytogenes can be protected from cleaning and sanitizing activities and persist in the environment over longer periods. This leads to food safety concerns and presents a serious threat to the viability of tree fruit packing businesses as well as public health.

III. Objectives

The objective is to better under the effectiveness of different combinations of cleaning and sanitizing practices in terms of inactivation of environmental microbiota (biofilms) and L.

monocytogenes is needed to develop evidence-based recommendations for cleaning/sanitizing procedures for the industry. The contractor shall assess the effectiveness of multiple combinations of physical and chemical cleaning and sanitizing procedures in removal of microbiome (biofilms) and inactivation of L. monocytogenes (Obj. 1). FDA will conduct this applied research project in collaboration with academia and our industry partners to help tree fruit packers maintain viable https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-019-0726-2 https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-019-0726-2 businesses and improve public health by improving tree fruit safety through effective control of L.

monocytogenes in packing facilities. Recently, FDA, in collaboration with the Pennsylvania State University research teams have sequenced three of the metagenomic samples from our published study using a Nanopore MinION to assess whether (i) Listeria monocytogenes can be detected directly in metagenomic DNA without enrichment or PCR amplification, (ii) whether Nanopore results are consistent with the 16S rRNA amplicon sequencing and (iii) whether we can obtain greater taxonomic resolution when characterizing environmental microbiota. We were able to detect L. monocytogenes directly in all three metagenomic DNA extracts, obtain taxonomic composition consistent with 16S rRNA amplicon-inferred composition, and were able to identify specific species of Pseudomonas that have been previously confidently identified only at a family level.

FDA researchers propose to build on these findings and utilize Nanopore sequencing in the current study (in Obj. 2) to characterize microbiota (including presence of L. monocytogenes) in a subset of samples collected within the frame of the Obj. 1. Furthermore, the contractor shall conduct comparative microbiome analyses to compare the performance of microbiome characterization using Nanopore and amplicon sequencing, as well as to evaluate the effect of different amplicon data analyses methods on the results and interpretation of the results.

IV. Specific Tasks

The goals of this study shall be achieved through the following specific objectives:

Obj. 1: Evaluate the effectiveness of different cleaning and sanitizing procedures in reduction of environmental microbiota and Listeria monocytogenes.

The contractor shall evaluate the effect of four different combinations of commonly used or recommended cleaners (C) and sanitizers (S) on the (i) the concentration of total aerobic mesophilic microorganisms, (ii) microbial community composition, and (iii) concentration of Listeria monocytogenes. The contractor shall collect 5 samples pre- and 5 samples post-cleaning and sanitizing, in three facilities, assessing 4 combination of cleaners and sanitizers, resulting in a total of 120 samples.

Combination 1: C1 + S1 (3 facilities, 10 samples per facility – 5 pre and 5 post cleaning and sanitation), n = 30;

Combination 2: C1 + S2 (3 facilities, 10 samples per facility – 5 pre and 5 post cleaning and sanitation), n = 30;

Combination 3: C2 + S1 (3 facilities, 10 samples per facility – 5 pre and 5 post cleaning and sanitation), n = 30;

Combination 4: C2 + S2 (3 facilities, 10 samples per facility – 5 pre and 5 post cleaning and sanitation), n = 30.

Each sample will be processed to (i) determine the aerobic plate count (APC), (ii) quantify L.

monocytogenes using and MPN method with media recommended by the FDA BAM, and (iii) characterize the microbiota using 16S rRNA V4 and ITS2 sequencing. Thirty samples collected pre- and post- most effective cleaning and sanitizing protocol will be additionally sequenced using a shotgun metagenomics approach with Nanopore MinION.

Obj. 2: Compare the microbial communities’ composition and ability to detect L.

monocytogenes using shotgun and amplicon sequencing

The shotgun metagenomic sequences obtained using MinION sequencing and amplicon sequences obtained using Illumina sequencing will be processed using separate pipelines to determine taxonomic composition of tested samples. Taxonomic composition will then be compared to assess the congruency of the results. Furthermore, the quantity of L. monocytogenes reads detected using Nanopore sequencing will be compared with the concentration of cultured L. monocytogenes as determined using the MPN method to assess the sensitivity of Nanopore sequencing for direct detection of L. monocytogenes.

V. Deliverables

• Contractor shall deliver:

1. Surveillance data on the aerobic plate count (APC) and quantitative data (using MPN method with media recommended by the FDA BAM) on L. monocytogenes concentrations for 120 samples collected as specified under the objective 1.

2. Results characterizing the microbiota using 16S rRNA V4 and ITS2 sequencing for 120 samples collected as specified under the objective 1

3. Results characterizing the microbiota using a shotgun metagenomics approach with Nanopore MinION for 30 samples collected as specified under the objective 1.

4. Results on the comparison of the microbial communities’ composition and ability to detect L.

monocytogenes using shotgun and amplicon sequencing, as specified under the objective 2.

VI. Travel

A total of 8 trips to collect samples from three tree fruit packing facilities in the state of Pennsylvania (~250 miles per trip), resulting in ~2,000 miles, and ~8 overnight stays in a hotel.

VII. Place of Performance

All laboratory research tasks shall be performed at the Pennsylvania State University in 437 Rodney A.

Erickson Food Science Building University Park, PA. Surveillance sampling shall be conducted at three tree fruit commercial packing facilities in the state of Pennsylvania.

VIII. Period of Performance

The period of performance shall be from August 01, 2020 to August 01, 2021.