Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
(N/A)
Non Technical Summary
Nationwide shortages of infant formula reached more than 50%,with some regions experiencing 95% shortages by the end of May 2022.This impacted millions of infants, disproportionally from low-income households, in the U.S. who lost access to their primary or exclusive nutrition source. Increases in imports supported by the U.S. Food and Drug Administration (FDA) unintentionally exacerbated food security issues worldwide already strained by the COVID-19 pandemic.The shortage resulted from a single production plant suspending operations in the U.S. after four infants were hospitalized and two died from consuming infant formula contaminated withCronobacter sakazakii.Environmental testing revealed the presence ofC. sakazakiion non-food contact surfaces within the facility. Specific strains associated with infant illnesses were not recovered from the facility,underscoring the complex balance of protecting public health and food security.There is mounting evidence from food and healthcare systems that pathogens can exist for extended periods of time in low-moisture environments as dry or desiccated biofilms.Under low-moisture conditions, foodborne pathogenscan form or become embedded in biofilms on surfaces.These biofilms can persist on surfaces as dry surface biofilms (DSB), which are hardto detect and eradicate leading to cross-contamination of foods. We hypothesize that foodborne pathogens persist in DSB over time and the presence of other organisms enhances survivability. We also hypothesize that current sanitation practices in dry food environments may not be sufficient to eliminate persistent pathogens within DSB. Our objectives are to (i) develop DSB models of important foodborne pathogens,(ii) evaluate current LMF sanitation efficacy against these models, and (iii) characterize three-dimensional structure and gene expression profiles of DSB models to refine sanitation strategies. Outcomes from this work will elucidate strategies to eliminate and ideally prevent DSB in LMF processing facilities (e.g., infant formula).Infant formula shortages exposed U.S. food production and distribution system fragilities, underscoring a need for better strategies to prepare for and respond to public health and food security threats. It further underscores opportunities to better support women, children, families, and low-income communities. The work proposed here, positioned at the intersection of food safety and food security, will fill knowledge gaps, and investigate solutions with private sector partners who will directly use and benefit from the outcomes of the proposed objectives.
Animal Health Component
50%
Research Effort Categories
Basic
40%
Applied
50%
Developmental
10%
Goals / Objectives
While significant work has been done to understand biofilm formation of foodborne pathogens and their role in cross-contamination to foods, there is limited peer reviewed work on dry surface biofilms in low-moisture food environments, their stress tolerance, and vetted strategies for their elimination. Standard sanitation strategies for these dry environments may not be sufficient to eliminate dry surface biofilms. We hypothesize that C. sakazakii persists in DSB and the presence of other organisms (e.g., P. aeruginosa) enhances its survivability. We further hypothesize that current sanitation practices in LMF environments may not be sufficient to eliminate persistent pathogens within DSB models. The overall goal of this proposal is to understand foodborne pathogen survival in DSB and to determine if current sanitation practices control these particularly difficult contamination challenges or if new strategies are warranted.Objectives:Develop in vitro mono- and multi-species DSB models of C. sakazakii in combination with S. enterica serovar Typhimurium, L. monocytogenes, and P. aeruginosaEvaluate current LMF sanitation efficacy against mono- and multi-species DSBCharacterize three-dimensional structural and gene expression profiles of DSB models to refine sanitation strategies
Project Methods
Objective 1. Develop in vitro desiccated mono- and multi-species biofilm models ofC. sakazakiiin combination withS. entericaserovar Typhimurium, L. monocytogenes,andP. aeruginosa. A CDC bioreaction assembly consisting of a one-liter glass vessel with polyethylene top, a stir plate assembly, eight polypropylene rods that can hold three coupons each, for a total of 24 borosilicate glass coupons will be used to develop biofilms. A sterilized CBR will be inoculated with one ml of approximately 107CFU overnight culture of each strain in a 500 ml batch phase (i.e., 0.3 or 3 gTryptic Soy Broth (TSB)per 1L deionized sterile water). The reactor baffle speed will be set at 60±5 rpm (Gram+) or 125±5 rpm (Gram-) at near room temperature i.e., 21±2°C. After 24 hrs, continuous phase (100 mg or 1 g TSB per 1L) will be initiated by connecting silicone tubing from a carboy containing continuous stir tank reactor (CSTR) growth medium (100 mg/L TSB or 1 g/L TSB) to the reactor. Growth media will be pumped into the reactor using a peristaltic pump (Cole-Palmer, Barrington, IL) to achieve a final flow rate of approximately 11.7 ml/min for 24±2 hrs; waste will be collected in a carboy.As funding permits or with supplementary funds from other sources, we will strive to include other materials beyond glass, such as stainless steel and othergrowth mediums, such as milk powder. All experiments will be done in triplicate. Mean bacterial log10 densities will be calculated based on CFU counts from wet and dry surface biofilm coupons. Average bacterial log10 densities will be compared for significant statistical differences using the least squares method of PROC GLM to fit linear models (α = 0.05). Significant differences between group means of bacterial log10 densities per dry time will also be compared pairwise using Tukey adjustments SAS version 9.4 (SAS Institute, Cary, NC) for all statistical analysis.Objective 2. Evaluate current LMF sanitation efficacy against mono- and multi-species DSB.Alcohol-based sanitizers with LMF environment usage claims and microfiber swabs will be tested against dry surface biofilm models refined in Obj. 1. using EPA biofilm disinfection efficacy testing protocols. Briefly, four ml of sanitizer at label-defined time and concentration or PBS in case of post-microfiber swabbing, will be used to treat coupons (n=3) at room temperature. Controls will be treated with four ml of PBS with a one-min contact time. Following treatment, sanitizers will be neutralized with 36 ml BD Difco neutralizing buffer solution (Fisher scientific, USA). Mean log10 reductions resulting from sanitizer treated or wiped coupons will be calculated and used for statistical analyses. Specifically, mean bacterial log10 densities per coupon will be calculated both for control (PBS treated) and microfiber swabbed or sanitizer treated coupons. Mean log10 densities per coupon will be normalized against the mean log10 densities of control coupons to determine log10 reductions. The least squares method of the PROC GLIMMIX procedure will be used to analyze and compare mean log10 reductions among the tested products (α = 0.05). The same test will be used to statistically compare mean log10 reductions at 24, 48, and 72 hrs of dehydration. Pair-wise comparisons among products, strains, and dry times will be completed with Tukey adjustments. All statistical procedures will be completed using SAS version 9.4 (SAS Institute, Cary, NC).Objective 3. Characterize three-dimensional structural and gene expression profiles of DSB to refine sanitation strategies. Biofilm fluorescent labeling and confocal laser scanning microscopy: DSB refined in Obj. 1 will be aseptically transferred to microtiter plates (Corning, USA). Each coupon will be fixed with 4% methanol-free solution of formaldehyde (Fisher Scientific, USA) for at least 15 min at room temperature. Coupons will immunostained and three-dimensional structurally characterized using CLSM. Results will also be validated by light microscopic techniques using Giemsa staining30 and crystal violet staining27 of the coupons. Work will be conducted in triplicate).EPS Staining: DSB coupons will be stained with SYTO 9 and Alexa Fluor 647-dextran conjugate. Polysaccharides will be labeled with Alexa Fluor 647-dextran conjugate [Molecular Probes, Invitrogen Corp., Carlsbad, CA, USA] and bacterial cells will be labeled by SYTO 9 green, fluorescent nucleic acid stain [Molecular Probes, Invitrogen Corp., Carlsbad, CA, USA).Live/dead staining: DSB coupons will be stained using the BacLight live/dead bacterial viability kit [Molecular Probes, Eugene, USA] as detailed in a previous study.14 Live bacteria cells will fluoresce green with SYTO 9, while cells with compromised membranes will stain red with propidium iodide dye; this will be visualized with CLSM [Leica, Wetzlar, Germany]. At least three coupons for each group will be examined and the tests will be repeated three times.RNA sequencing: Coupons will be fixed with RNAprotect (Qiagen, Hilden, Germany). Cells will be released from the coupon as described in Obj. 1. Cells will be pelleted, RNA extracted, and library prepared as outlined in Boonmee et al.82 after DNase treatment using TURBO DNA-freeTM DNase treatment kit [Invitrogen, USA]. Ribo-Zero rRNA removal kits (Illumina, San Diego, CA) will be used to deplete bacterial ribosomal RNA. mRNA will be reverse transcribed to cDNA, then purified using Agencourt AMPure XP kit [Beckman Coulter Inc., Brea, CA]. Sample quality and quantity will be assessed on a 2100 bioanalyzer [Agilent Technology, Santa Clara, CA]. cDNA sequencing libraries will be constructed using ScriptSeq v2 RNA-seq library preparation kit [Epicentre, Madison, WI, USA] and sequenced on an Illumina HiSeq2500 at Purdue University. RNA-sequencing reads will be processed and mapped to closed genomes and differentially expressed genes (DEGs) will be determined. All work will be performed in triplicate.