The Use of Indicators and Surrogate Microorganisms for the Evaluation of Pathogens in Fresh and Fresh‐Cut Produce

dEnterococcus faecium NRRL B-2354 is a surrogate microorganism used in place of pathogens for validation of thermal processing technologies and systems. We evaluated the safety of strain NRRL B-2354 based on its genomic and functional characteristics. The genome of E. faecium NRRL B-2354 was sequenced and found to comprise a 2,635,572-bp chromosome and a 214,319-bp megaplasmid. A total of 2,639 coding sequences were identified, including 45 genes unique to this strain. Hierarchical clustering of the NRRL B-2354 genome with 126 other E. faecium genomes as well as pbp5 locus comparisons and multilocus sequence typing (MLST) showed that the genotype of this strain is most similar to commensal, or community-associated, strains of this species. E. faecium NRRL B-2354 lacks antibiotic resistance genes, and both NRRL B-2354 and its clonal relative ATCC 8459 are sensitive to clinically relevant antibiotics. This organism also lacks, or contains nonfunctional copies of, enterococcal virulence genes including acm, cyl, the ebp operon, esp, gelE, hyl, IS16, and associated phenotypes. It does contain scm, sagA, efaA, and pilA, although either these genes were not expressed or their roles in enterococcal virulence are not well understood. Compared with the clinical strains TX0082 and 1,231,502, E. faecium NRRL B-2354 was more resistant to acidic conditions (pH 2.4) and high temperatures (60°C) and was able to grow in 8% ethanol. These findings support the continued use of E. faecium NRRL B-2354 in thermal process validation of food products.

Section: Discussionmentioning

confidence: 99%

Safety of the Surrogate Microorganism Enterococcus faecium NRRL B-2354 for Use in Thermal Process Validation

Kopit

Kim

Siezen

et al. 2014

“…It should be predictable, easily detected, and decontaminated without jeopardizing industrial equipment or natural environments (11). In these cases, nonviable surrogates, such as polyvinyl spheres, are useful because their persistence poses no concern for infection of laboratory personnel, environmental pollution, or mutation.…”

Section: Conceptual Decision Frameworkmentioning

confidence: 99%

“…These inactivation models can predict the survival of pathogens in the environment for given temperatures (48). High temperatures denature proteins and nucleic acids, while only a few possibilities exist for resistant organisms, such as bacterial spores, parvovirus, and Bacillus stearothermophilus spores (11,51). Additionally, the pH and cation/anion balance associated with the external environment can stabilize or destabilize proteins, a process which can therefore increase or decrease resistance to thermal inactivation (17).…”

Section: Conceptual Decision Frameworkmentioning

confidence: 99%

Criteria for Selection of Surrogates Used To Study the Fate and Control of Pathogens in the Environment

Sinclair

Rose

Hashsham

et al. 2012

132

106

e This article defines the term surrogate as an organism, particle, or substance used to study the fate of a pathogen in a specific environment. Pathogenic organisms, nonpathogenic organisms, and innocuous particles have been used as surrogates for a variety of purposes, including studies on survival and transport as well as for method development and as "indicators" of certain conditions. This article develops a qualitative surrogate attribute prioritization process and allows investigators to select a surrogate by systematically detailing the experimental process and prioritizing attributes. The results are described through the use of case studies of various laboratories that have used this process. This article also discusses the history of surrogate and microbial indicator use and outlines the method by which surrogates can be used when conducting a quantitative microbial risk assessment. The ultimate goal of selecting a sufficiently representative surrogate is to improve public health through a health-based risk assessment framework. Under-or overestimating the resistance, inactivation, or movement may negatively impact risk assessments that, in turn, will impact health assessments and estimated safety levels. Reducing uncertainty in a risk assessment is one of the objectives of using surrogates and the ultimate motive for any experiment investigating potential exposure of a pathogen.

“…However, field experiments with virulent human pathogens are unlikely to receive regulatory approval due to the obvious risks associated with environmental exposure. Surrogate organisms have been used to better understand how human pathogens survive in environments and respond to mitigation treatments (15)(16)(17). Surrogates are deliberately introduced in order to assess the effectiveness of disinfection, sanitation, or other treatments (17).…”

Section: Importancementioning

confidence: 99%

Development of an Avirulent Salmonella Surrogate for Modeling Pathogen Behavior in Pre- and Postharvest Environments

Moraes

Chapin

Ginn³

et al. 2016

Recurrent outbreaks of bacterial gastroenteritis linked to the consumption of fresh fruits and vegetables highlight the paucity of understanding of the ecology of Salmonella enterica under crop production and postharvest conditions. These gaps in knowledge are due, at least in part, to the lack of suitable surrogate organisms for studies for which biosafety level 2 is problematic. Therefore, we constructed and validated an avirulent strain of Salmonella enterica serovar Typhimurium. The strain lacks major Salmonella pathogenicity islands SPI-1, SPI-2, SPI-3, SPI-4, and SPI-5 as well as the virulence plasmid pSLT. Deletions and the absence of genomic rearrangements were confirmed by genomic sequencing, and the surrogate behaved like the parental wildtype strain on selective media. A loss-of-function (phoN) selective marker allowed the differentiation of this strain from wildtype strains on a medium containing a chromogenic substrate for alkaline phosphatase. Lack of virulence was confirmed by oral infection of female BALB/c mice. The strain persisted in tomatoes, cantaloupes, leafy greens, and soil with the same kinetics as the parental wild-type and selected outbreak strains, and it reached similar final population levels. The responses of this strain to heat treatment and disinfectants were similar to those of the wild type, supporting its potential as a surrogate for future studies on the ecology and survival of Salmonella in production and processing environments. IMPORTANCEThere is significant interest in understanding the ecology of human pathogens in environments outside of their animal hosts, including the crop production environment. However, manipulative field experiments with virulent human pathogens are unlikely to receive regulatory approval due to the obvious risks. Therefore, we constructed an avirulent strain of S. enterica serovar Typhimurium and characterized it extensively. O utbreaks of human disease caused by nontyphoidal Salmonella enterica have been linked to sprouts, fruits, vegetables, and nuts that were contaminated at various points in the production cycle (1-8). Generic Escherichia coli indicator levels are currently monitored to ensure the safety of produce; however, the persistence of Salmonella on the surfaces of peaches, cantaloupes, lettuce, and bell peppers has been shown to be very different from that of E. coli (9, 10). In general, Salmonella survives better outside of its animal hosts than E. coli (11) and can persist in processed organic waste and amended soils better than fecal coliforms, although its persistence appears to be strain and/or site dependent (12-14).There is significant interest in understanding the ecology of specific pathogens in the environment outside of their animal hosts. However, field experiments with virulent human pathogens are unlikely to receive regulatory approval due to the obvious risks associated with environmental exposure. Surrogate organisms have been used to better understand how human pathogens survive in environments and respond to...