Phenotypic Characteristics That May Contribute to Persistence of Salmonella Strains in the Pistachio Supply Chain.

Salmonella enterica subsp. enterica strain diversity in California pistachios is limited; some strains have persisted in the pistachio supply chain for ≥10 years. Representative isolates of six persistent strains and three sporadic strains isolated from California pistachios were selected to evaluate copper resistance, growth in pistachio hull slurry, biofilm formation, desiccation tolerance, and survival during subsequent storage. The presence of a copper homeostasis and silver-resistance island sequence in three of the persistent strains was associated with an increase in tolerance to CuSO4 from 7.5 mM to 15 mM under anaerobic but not aerobic conditions; all isolates were resistant to ≥120 mM Cu-EDTA under both anerobic and aerobic conditions. When inoculated into pistachio hull slurry at 2.75 ± 0.04 log CFU/mL and incubated at 30 °C, the populations of Salmonella Enteritidis strain A (sporadic) increased to significantly lower levels than the other strains at 16, 20, 24, and 28 h but not at 40 and 48 h. Maximum populations of 8.70-8.85 log CFU/mL were observed for all strains at ≥40 h of incubation. All nine Salmonella strains produced weak to strong biofilms after 4 days at 25 °C; seven strains, including two sporadic strains, produced moderate biofilms, and Salmonella Liverpool strain A (persistent) produced a strong biofilm. The rdar+ and rdar- morphotypes were observed in both persistent and sporadic Salmonella strains. Population declines of 5.03 log were observed for Salmonella Enteritidis strain A within 18 h of drying on filter paper whereas reductions of 0.50-1.25 log were observed for the other eight Salmonella strains. Population reductions (3.98-5.12 log) of these eight strains were not significantly different after storage at 25 ± 1 °C and 35% relative humidity for 50 days. The phenotypic characteristics evaluated here do not independently account for the persistence of a small number of Salmonella strains associated with the California pistachio production chain.

Characterizing the Genetic Diversity of Salmonella Isolated from U.S. Raw Inshell Pistachios Using Whole Genome Sequencing.

The genetic diversity of 169 Salmonella isolates from pistachios collected from California storage silos during the 2010, 2011, and 2012 harvests (silo survey isolates) was determined by analyzing the whole genome sequence data using the CFSAN SNP pipeline developed by the U.S. Food and Drug Administration's Center for Food Safety and Applied Nutrition. Salmonella isolates clustered by serovars Agona, Enteritidis, Montevideo, Sandiego, Senftenberg, Liverpool, Tennessee, and Worthington in the phylogenetic tree. Within each serovar, isolates grouped into one or two clusters (≤14 SNPs). Two distinct clusters (>14 SNPs; A and B) were identified for Salmonella Enteritidis, Montevideo, and Liverpool for a total of 11 unique strains. Sequences of representative silo survey isolates clustered with sequences of Salmonella strains isolated from U.S. pistachio-associated samples collected between 2008 and 2018 available on the National Center for Biotechnology Information database, and, in all but two cases, not with sequences of Salmonella strains recovered from raw California almonds from 2001 through 2013. The genomic evidence suggests that strains of Salmonella Agona, Liverpool Cluster A, Montevideo Clusters A and B, Senftenberg, and Worthington have persisted in the California pistachio environment for ≥3 years and some of these strains have been reported exclusively in association with pistachios.

Fate of foodborne pathogens during soaking and drying of walnuts.

Walnuts are among the most popular tree nuts that are soaked at home. Recipes for preparing soaked walnut kernels from online blogs (n = 71) and YouTube videos (n = 29) were reviewed to identify typical consumer handling practices that were then used to determine the fate of foodborne pathogens during soaking and subsequent drying of walnut kernels. Individual five-strain cocktails of rifampin-resistant Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella, grown on agar plates and diluted in water, were inoculated onto walnuts and then dried. Inoculated walnuts were added to sterile water at a ratio of 1:4 (w/v), held at 15, 18, or 22°C for up to 24 h, and then dried at 64°C for up to 24 h (for Salmonella-inoculated walnuts). Pathogen populations during soaking and drying were enumerated on tryptic soy agar with rifampin and on CHROM agar. Initial walnut moisture was ∼4%, increased to ∼30% at 8 and 24 h of soaking and then decreased during drying to ∼4% at 6 h and <1% after 24 h. Initial E. coli, L. monocytogenes, and Salmonella populations were ∼1.0, ∼1.5, and 1.0-2.5 log CFU/g, respectively, after inoculation and drying. No significant (P > 0.05) increase in populations was observed after 24 h at 15 and 18°C or after 12 h at 22°C. Significant increases of 1.9-3.0, 1.2-2.1, and 1.8 log CFU/g for E. coli, L. monocytogenes, and Salmonella, respectively, were observed after 24 h of soaking at 22°C. Growth rates of 0.19, 0.093, and 0.16 log CFU/sample per h, respectively, were observed. Lag times of 8.8 and 11 h at 22°C were determined for E. coli and Salmonella, respectively. Populations of Salmonella declined by 1.04 log CFU/g over 12 h of drying; further significant (P < 0.05) decreases were not observed at 24 h. To limit food safety risks in soaked walnuts, educational materials should emphasize sourcing treated walnuts, kitchen sanitation, hygiene measures, and soaking at cooler temperatures or for shorter times at ambient temperatures.

Humans and Hoofed Livestock Are the Main Sources of Fecal Contamination of Rivers Used for Crop Irrigation: A Microbial Source Tracking Approach.

Freshwater bodies receive waste, feces, and fecal microorganisms from agricultural, urban, and natural activities. In this study, the probable sources of fecal contamination were determined. Also, antibiotic resistant bacteria (ARB) were detected in the two main rivers of central Chile. Surface water samples were collected from 12 sampling sites in the Maipo (n = 8) and Maule Rivers (n = 4) every 3 months, from August 2017 until April 2019. To determine the fecal contamination level, fecal coliforms were quantified using the most probable number (MPN) method and the source of fecal contamination was determined by Microbial Source Tracking (MST) using the Cryptosporidium and Giardia genotyping method. Separately, to determine if antimicrobial resistance bacteria (AMB) were present in the rivers, Escherichia coli and environmental bacteria were isolated, and the antibiotic susceptibility profile was determined. Fecal coliform levels in the Maule and Maipo Rivers ranged between 1 and 130 MPN/100-ml, and 2 and 30,000 MPN/100-ml, respectively. Based on the MST results using Cryptosporidium and Giardia host-specific species, human, cattle, birds, and/or dogs hosts were the probable sources of fecal contamination in both rivers, with human and cattle host-specific species being more frequently detected. Conditional tree analysis indicated that coliform levels were significantly associated with the river system (Maipo versus Maule), land use, and season. Fecal coliform levels were significantly (p < 0.006) higher at urban and agricultural sites than at sites immediately downstream of treatment centers, livestock areas, or natural areas. Three out of eight (37.5%) E. coli isolates presented a multidrug-resistance (MDR) phenotype. Similarly, 6.6% (117/1768) and 5.1% (44/863) of environmental isolates, in Maipo and Maule River showed and MDR phenotype. Efforts to reduce fecal discharge into these rivers should thus focus on agriculture and urban land uses as these areas were contributing the most and more frequently to fecal contamination into the rivers, while human and cattle fecal discharges were identified as the most likely source of this fecal contamination by the MST approach. This information can be used to design better mitigation strategies, thereby reducing the burden of waterborne diseases and AMR in Central Chile.

Prevalence, Persistence, and Diversity of Listeria monocytogenes and Listeria Species in Produce Packinghouses in Three U.S. States.

ABSTRACT: Listeria monocytogenes has emerged as a food safety concern for several produce commodities. Although L. monocytogenes contamination can occur throughout the supply chain, contamination from the packinghouse environment represents a particular challenge and has been linked to outbreaks and recalls. This study aimed to investigate the prevalence, persistence, and diversity of L. monocytogenes and other species of Listeria in produce packinghouses. A longitudinal study was performed in 11 packinghouses (whose commodities included microgreen, peach, apple, tomato, broccoli, cauliflower, and cucumber) in three U.S. states. In each packinghouse, 34 to 47 sites representing zones 2 to 4 were selected and swabbed. Packinghouses were visited four times over the packing season, and samples were tested for Listeria by following the U.S. Food and Drug Administration's Bacteriological Analytical Manual methods. Presumptive Listeria-positive isolates were confirmed by PCR. Species and allelic type (AT) were identified by sigB sequencing for up to eight isolates per sample. Among 1,588 samples tested, 50 (3.2%), 42 (2.7%), and 10 (0.6%) samples were positive for L. monocytogenes only, Listeria spp. (excluding L. monocytogenes) only, and both L. monocytogenes and Listeria spp., respectively. Five species of Listeria (L. monocytogenes, L. innocua, L. seeligeri, L. welshimeri, and L. marthii) were identified, and L. monocytogenes was the most prevalent species. The 102 Listeria-positive samples yielded 128 representative isolates (i.e., defined as isolates from a given sample with a different AT). Approximately 21% (21 of 102) of the Listeria-positive samples contained two or more ATs. A high AT diversity (0.95 Simpson's diversity index) was observed among Listeria isolates. There were three cases of L. monocytogenes or Listeria spp. repeated isolation (site testing positive at least twice) based on AT data. Data from this study also support the importance of drain and moisture management, because Listeria were most prevalent in samples collected from drain, cold storage, and wet nonfood contact surface sites.