Integrative Assessment of Reduced Listeria monocytogenes Susceptibility to Benzalkonium Chloride in Produce Processing Environments.

For decades, quaternary ammonium compounds (QAC)-based sanitizers have been broadly used in food processing environments to control foodborne pathogens such as Listeria monocytogenes. Still, there is a lack of consensus on the likelihood and implication of reduced Listeria susceptibility to benzalkonium chloride (BC) that may emerge due to sublethal exposure to the sanitizers in food processing environments. With a focus on fresh produce processing, we attempted to fill multiple data and evidence gaps surrounding the debate. We determined a strong correlation between tolerance phenotypes and known genetic determinants of BC tolerance with an extensive set of fresh produce isolates. We assessed BC selection on L. monocytogenes through a large-scale and source-structured genomic survey of 25,083 publicly available L. monocytogenes genomes from diverse sources in the United States. With the consideration of processing environment constraints, we monitored the temporal onset and duration of adaptive BC tolerance in both tolerant and sensitive isolates. Finally, we examined residual BC concentrations throughout a fresh produce processing facility at different time points during daily operation. While genomic evidence supports elevated BC selection and the recommendation for sanitizer rotation in the general context of food processing environments, it also suggests a marked variation in the occurrence and potential impact of the selection among different commodities and sectors. For the processing of fresh fruits and vegetables, we conclude that properly sanitized and cleaned facilities are less affected by BC selection and unlikely to provide conditions that are conducive for the emergence of adaptive BC tolerance in L. monocytogenes. IMPORTANCE Our study demonstrates an integrative approach to improve food safety assessment and control strategies in food processing environments through the collective leveraging of genomic surveys, laboratory assays, and processing facility sampling. In the example of assessing reduced Listeria susceptibility to a widely used sanitizer, this approach yielded multifaceted evidence that incorporates population genetic signals, experimental findings, and real-world constraints to help address a lasting debate of policy and practical importance.

Soil Bacteria in Urban Community Gardens Have the Potential to Disseminate Antimicrobial Resistance Through Horizontal Gene Transfer.

Fifteen soil and 45 vegetable samples from Detroit community gardens were analyzed for potential antimicrobial resistance contamination. Soil bacteria were isolated and tested by antimicrobial susceptibility profiling, horizontal gene transfer, and whole-genome sequencing. High-throughput 16S rRNA sequencing analysis was conducted on collected soil samples to determine the total bacterial composition. Of 226 bacterial isolates recovered, 54 were from soil and 172 from vegetables. A high minimal inhibitory concentration (MIC) was defined as the MIC greater than or equal to the resistance breakpoint of Escherichia coli for Gram-negative bacteria or Staphylococcus aureus for Gram-positive bacteria. The high MIC was observed in 63.4 and 69.8% of Gram-negative isolates from soil and vegetables, respectively, against amoxicillin/clavulanic acid, as well as 97.5 and 82.7% against ampicillin, 97.6 and 90.7% against ceftriaxone, 85.4 and 81.3% against cefoxitin, 65.8 and 70.5% against chloramphenicol, and 80.5 and 59.7% against ciprofloxacin. All Gram-positive bacteria showed a high MIC to gentamicin, kanamycin, and penicillin. Forty of 57 isolates carrying tetM (70.2%) successfully transferred tetracycline resistance to a susceptible recipient via conjugation. Whole-genome sequencing analysis identified a wide array of antimicrobial resistance genes (ARGs), including those encoding AdeIJK, Mex, and SmeDEF efflux pumps, suggesting a high potential of the isolates to become antimicrobial resistant, despite some inconsistency between the gene profile and the resistance phenotype. In conclusion, soil bacteria in urban community gardens can serve as a reservoir of antimicrobial resistance with the potential to transfer to clinically important pathogens, resulting in food safety and public health concerns.

Global spread of Salmonella Enteritidis via centralized sourcing and international trade of poultry breeding stocks.

A pandemic of Salmonella enterica serotype Enteritidis emerged in the 1980s due to contaminated poultry products. How Salmonella Enteritidis rapidly swept through continents remains a historical puzzle as the pathogen continues to cause outbreaks and poultry supply becomes globalized. We hypothesize that international trade of infected breeding stocks causes global spread of the pathogen. By integrating over 30,000 Salmonella Enteritidis genomes from 98 countries during 1949-2020 and international trade of live poultry from the 1980s to the late 2010s, we present multifaceted evidence that converges on a high likelihood, global scale, and extended protraction of Salmonella Enteritidis dissemination via centralized sourcing and international trade of breeding stocks. We discovered recent, genetically near-identical isolates from domestically raised poultry in North and South America. We obtained phylodynamic characteristics of global Salmonella Enteritidis populations that lend spatiotemporal support for its dispersal from centralized origins during the pandemic. We identified concordant patterns of international trade of breeding stocks and quantitatively established a driving role of the trade in the geographic dispersal of Salmonella Enteritidis, suggesting that the centralized origins were infected breeding stocks. Here we demonstrate the value of integrative and hypothesis-driven data mining in unravelling otherwise difficult-to-probe pathogen dissemination from hidden origins.

Survival and transcriptomic response of Salmonella enterica on fresh-cut fruits.

Salmonella enterica is frequently implicated in foodborne disease outbreaks associated with fresh-cut fruits. In the U.S., more than one third of fruit-related outbreaks have been linked to two S. enterica serotypes Newport and Typhimurium. Approximately 80% of fruit-related human salmonellosis cases were associated with tomatoes, cantaloupes and cucumbers. In this study, we investigated the population dynamics of S. Newport and S. Typhimurium on fresh-cut tomato, cantaloupe, cucumber and apple under short-term storage conditions. We further compared the transcriptomic profiles of a S. Newport strain on fresh-cut tomato and cantaloupe using high-throughput RNA-seq. We demonstrated that both S. enterica Newport and Typhimurium survived well on various fresh-cut fruit items under refrigeration storage conditions, independent of inoculation levels. However, S. enterica displayed variable survival behaviors on different types of fruits. For example, at 7 d storage, the population of S. enterica reduced less than 0.2 log (p > 0.05) on fresh-cut tomato and cantaloupe, in contrast to ~0.5 log (p < 0.05) on cucumber and apple. RNA-seq analysis suggested that S. enterica mediates its survival on fresh-cut fruits through differentially regulating genes involved in specific carbon utilization and metabolic pathways. Several known bacterial virulence factors (e.g., pag gene) were found to be differentially regulated on fresh-cut tomato and cantaloupe, suggesting a link between the events of food contamination and subsequent human infection. Findings from this study contribute to a better understanding of S. enterica survival mechanisms on fresh-cut produce.

Mechanisms of Salmonella Attachment and Survival on In-Shell Black Peppercorns, Almonds, and Hazelnuts.

Salmonella enterica subspecies I (ssp 1) is the leading cause of hospitalizations and deaths due to known bacterial foodborne pathogens in the United States and is frequently implicated in foodborne disease outbreaks associated with spices and nuts. However, the underlying mechanisms of this association have not been fully elucidated. In this study, we evaluated the influence of storage temperature (4 or 25°C), relative humidity (20 or 60%), and food surface characteristics on the attachment and survival of five individual strains representing S. enterica ssp 1 serovars Typhimurium, Montevideo, Braenderup, Mbandaka, and Enteritidis on raw in-shell black peppercorns, almonds, and hazelnuts. We observed a direct correlation between the food surface roughness and S. enterica ssp 1 attachment, and detected significant inter-strain difference in survival on the shell surface under various storage conditions. A combination of low relative humidity (20%) and ambient storage temperature (25°C) resulted in the most significant reduction of S. enterica on shell surfaces (p < 0.05). To identify genes potentially associated with S. enterica attachment and survival on shell surfaces, we inoculated a library of 120,000 random transposon insertion mutants of an S. Enteritidis strain on almond shells, and screened for mutant survival after 1, 3, 7, and 14 days of storage at 20% relative humidity and 25°C. Mutants in 155 S. Enteritidis genes which are involved in carbohydrate metabolic pathways, aerobic and anaerobic respiration, inner membrane transport, and glutamine synthesis displayed significant selection on almond shells (p < 0.05). Findings of this study suggest that various food attributes, environmental factors, and an unexpectedly complex metabolic and regulatory network in S. enterica ssp 1 collectively contribute to the bacterial attachment and survival on low moisture shell surface, providing new data for the future development of knowledge-based intervention strategies.

Glove-mediated transfer of Listeria monocytogenes on fresh-cut cantaloupe.

The common use of gloves in retail practices represents a potential route for cross contamination of foodborne pathogens in fresh-cut produce. Using fresh-cut cantaloupe as a food model, we investigated factors that may influence glove-mediated cross contamination by Listeria monocytogenes and developed mathematical models to illustrate the patterns of transfer during fresh-cutting practices. Contact time (2, 5, 10 s), contact pressure (0.05, 0.18, 0.37 psi), and glove type (nitrile, polyvinyl chloride, polyethylene) did not have a significant effect on transfer of L. monocytogenes from cantaloupe rind to flesh, or from flesh to flesh. However, glove type appeared to affect L. monocytogenes transfer from the stem scar tissue to cantaloupe flesh (P = 0.0371). Transfer from rind pieces that had been washed with water was significantly higher than transfer from pieces that had not been washed (P = 0.0006). Predictive modeling and experimental validation suggested that transfer of L. monocytogenes on cantaloupe flesh persists over 85 pieces through consecutive contacts with a gloved hand. Findings of the study provide new scientific data to aid researchers, retailers, and caterers in safety risk assessments of fresh-cut practices used to prepare cantaloupes and other produce items.

Case study on the soil antibiotic resistome in an urban community garden.

Urban agricultural soils can be an important reservoir of antibiotic resistance, and have great food safety and public health indications. This study investigated antibiotic-resistant bacteria and antibiotic resistance genes in urban agricultural soils using phenotypic and metagenomic tools. In total, 207 soil bacteria were recovered from 41 soil samples collected from an urban agricultural garden in Detroit, MI, USA. The most prevalent antibiotic resistance phenotype demonstrated by Gram-negative bacteria was resistance to ampicillin (94.2%), followed by chloramphenicol (80.0%), cefoxitin (79.5%), gentamicin (78.4%) and ceftriaxone (71.1%). All Gram-positive bacteria were resistant to gentamicin, kanamycin and penicillin. Genes encoding resistance to quinolones, ß-lactams and tetracyclines were the most prevalent and abundant in the soil. qepA and tetA, both encoding efflux pumps, predominated in the quinolone and tetracycline resistance genes tested, respectively. Positive correlation (P<0.05) was identified among groups of antibiotic resistance genes, and between antibiotic resistance genes and metal resistance genes. The data demonstrated a diverse population of antibiotic resistance in urban agricultural soils. Phenotypic determination together with soil metagenomics proved to be a valuable tool to study the nature and extent of antibiotic resistance in the environment.

Increased water activity reduces the thermal resistance of Salmonella enterica in peanut butter.

Increased water activity in peanut butter significantly (P < 0.05) reduced the heat resistance of desiccation-stressed Salmonella enterica serotypes treated at 90 °C. The difference in thermal resistance was less notable when strains were treated at 126 °C. Using scanning electron microscopy, we observed minor morphological changes of S. enterica cells resulting from desiccation and rehydration processes in peanut oil.

Survival and heat resistance of Salmonella enterica and Escherichia coli O157:H7 in peanut butter.

Significant differences (P < 0.05) were found between the survival rates of Salmonella enterica and Escherichia coli O157:H7 in peanut butter with different formulations and water activity. High carbohydrate content in peanut butter and low incubation temperature resulted in higher levels of bacterial survival during storage but lower levels of bacterial resistance to heat treatment.