Lijun Hu

Prevalence and Level of Listeria monocytogenes in Ice Cream Linked to a Listeriosis Outbreak in the United States

A most-probable-number (MPN) method was used to enumerate Listeria monocytogenes in 2,320 commercial ice cream scoops manufactured on a production line that was implicated in a 2015 listeriosis outbreak in the United States. The analyzed samples were collected from seven lots produced in November 2014, December 2014, January 2015, and March 2015. L. monocytogenes was detected in 99% (2,307 of 2,320) of the tested samples (lower limit of detection, 0.03 MPN/g), 92% of which were contaminated at <20 MPN/g. The levels of L. monocytogenes in these samples had a geometric mean per lot of 0.15 to 7.1 MPN/g. The prevalence and enumeration data from an unprecedented large number of naturally contaminated ice cream products linked to a listeriosis outbreak provided a unique data set for further understanding the risk associated with L. monocytogenes contamination for highly susceptible populations.

Prevalence of Salmonella in Cashews, Hazelnuts, Macadamia Nuts, Pecans, Pine Nuts, and Walnuts in the United States

Nuts have been identified as a vector for salmonellosis. The objective of this project was to estimate the prevalence and contamination level of Salmonella in raw tree nuts (cashews, pecans, hazelnuts, macadamia nuts, pine nuts, and walnuts) at retail markets in the United States. A total of 3,656 samples of six types of tree nuts were collected from different types of retail stores and markets nationwide between October 2014 and October 2015. These samples were analyzed using a modified version of the Salmonella culture method from the U.S. Food and Drug Administrations Bacteriological Analytical Manual. Of the 3,656 samples collected and tested, 32 were culturally confirmed as containing Salmonella. These isolates represented 25 serotypes. Salmonella was not detected in pecans and in-shell hazelnuts. Salmonella prevalence estimates (and 95% confidence intervals) in cashews, shelled hazelnuts, pine nuts, walnuts, and macadamia nuts were 0.55% [0.15, 1.40], 0.35% [0.04, 1.20], 0.48% [0.10, 1.40], 1.20% [0.53, 2.40], and 4.20% [2.40, 6.90], respectively. The rates of Salmonella isolation from major or big chain supermarkets, small chain supermarkets, discount, variety, or drug stores, and online were 0.64% [0.38, 1.00], 1.60% [0.80, 2.90], 0.00% [0.00, 2.40], and 13.64% [2.90, 35.00], respectively (Cochran-Mantel-Haenszel test: P = 0.02). The rates of Salmonella isolation for conventional and organic nuts were not significantly different. Of the samples containing Salmonella, 60.7% had levels less than 0.003 most probable number (MPN)/g. The highest contamination level observed was 0.092 MPN/g. The prevalence and levels of Salmonella in these tree nut samples were comparable to those previously reported for similar foods.

Evaluation of Three Real-Time PCR Methods for Detection of Salmonella from Cloves

The purpose of the study was to evaluate three real-time PCR platforms for rapid detection of Salmonella from cloves and to compare three different DNA extraction methods. Six trials were conducted with two clove cultivars, Ceylon and Madagascar, and three Salmonella serotypes, Montevideo, Typhimurium, and Weltevreden. Each trial consisted of 20 test portions. The preenrichment cultures were used to perform PCR for comparison of the effectiveness of U.S. Food and Drug Administration, Pacific Regional Laboratory Southwest (FDA-PRLSW), Applied Biosystems Inc. (ABI) MicroSEQ, and GeneDisc platforms for detection of Salmonella. Three DNA extraction methods were used: standard extraction method for each PCR platform, boil preparation, and LyseNow food pathogen DNA extraction cards. The results from real-time PCR correlated well with FDA Bacteriological Analytical Manual culture assay results, with a wide range of cycle threshold (CT) values among the three PCR platforms for intended positive samples. The mean CT values for MicroSEQ (16.36 ± 2.78) were significantly lower than for PRLSW (20.37 ± 3.45) and GeneDisc (23.88 ± 2.90) (P < 0.0001). Pairwise comparisons between PCR platforms using different DNA extraction methods indicate that the CT values are inversely proportional to the relative DNA quantity (RDQ) yields by different platform-extraction combinations. The pairing of MicroSEQ and boil preparation generated the highest RDQ of 120 and the lowest average CT value of 14.48, whereas the pairing of GeneDisc and LyseNow generated the lowest RDQ of 0.18 and the highest average CT of 25.97. Boil preparation yielded higher RDQ than the other extraction methods for all three PCR platforms. Although the MicroSEQ platform generated the lowest CT values, its sensitivity was compromised by narrow separations between the positive and negative samples. The PRLSW platform generated the best segregation between positive and negative groups and is less likely to produce false results. In conclusion, FDA-PRLSW was the most efficient PCR assay for Salmonella detection, and boil preparation was the best method for DNA extraction.

Evaluation of 3M Molecular Detection System and ANSR Pathogen Detection System for rapid detection of Salmonella from egg products

&NA; Isothermal amplification assay is a novel simple detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to evaluate the effectiveness of the 3M Molecular Detection System (MDS) and ANSR Pathogen Detection System (PDS) for the detection of Salmonella in egg products as compared to the Food and Drug Administrations Bacteriological Analytical Manual (BAM) culture method and a modified culture method (3M MDS and ANSR PDS preferred method). Two Salmonella ser. Enteritidis (18579, PT4; CDC_2010K_1441, PT8), one Salmonella ser. Heidelberg (607310‐1), and one Salmonella ser. Typhimurium (0723) isolates were used in this study. Seven wet egg products and 13 dry egg products were inoculated with these strains individually at 1 to 5 CFU/25 g. One set of test portions was prepared following FDA BAM procedures [with lactose broth (LB) as pre‐enrichment broth]. Another set of test portions was prepared using buffered peptone water (BPW) as pre‐enrichment broth, as instructed by the 2 detection systems. Results from 3M MDS and ANSR PDS were 100% in agreement with their BPW‐based culture method results. When LB was used as pre‐enrichment broth, the number of Salmonella positive test portions (80 tested), identified with the BAM, 3M MDS, and ANSR PDS, were 63, 61, and 60, respectively. In conclusion, both 3M MDS and ANSR PDS Salmonella assays were as effective as their BPW based culture methods and were equivalent to the BAM culture method for the detection of Salmonella in egg products. These sensitive isothermal assays can be used as rapid detection tools for Salmonella in egg products provided that BPW is used as pre‐enrichment broth.

Development and Validation of a Cultural Method for the Detection and Isolation of Salmonella in Cloves

Detection of Salmonella in some spices, such as cloves, remains a challenge due to their inherent antimicrobial properties. The purpose of this study was to develop an effective detection method for Salmonella from spices using cloves as a model. Two clove varieties, Ceylon and Madagascar, were used in the study. Cloves were inoculated with Salmonella enterica subsp. enterica serotypes Montevideo, Typhimurium, or Weltevreden at about 1, 3, or 6 log CFU/25 g. Two test portion sizes, 10 and 25 g, were compared. After adding Trypticase soy broth (TSB) to the weighed cloves for preenrichment, three preenrichment methods were compared: cloves were left in the TSB for 24 h during preenrichment (PreE1), or the cloves-TSB mixture was shaken vigorously for 30 s (PreE2) or 60 s (PreE3), and the decanted material was transferred to a new bag for 24 h of preenrichment. The rest of the procedures were carried out according to the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM). At the low inoculation level (<1 log CFU/25 g), the detection rate was low across the three preenrichment methods, with the highest for PreE3 and lowest for PreE1. At the medium and high inoculation levels (3 and 6 log CFU/25 g), all samples from PreE2 and PreE3 were positive for Salmonella , whereas PreE1 produced only 12 positive samples from the 48 samples at the medium inoculation level and 38 positive samples from the 48 samples at the high inoculation level. Therefore, PreE3 with 25 g of cloves per sample was more effective than the other two tested methods. This newly designed method was then validated by comparing with the BAM method in six trials, with each trial consisting of 40 test samples. The results showed that PreE3 detected Salmonella from 88 of 120 inoculated test samples compared with only 31 positive from 120 test samples with the BAM method. Thus, our newly designed method PreE3 was more sensitive and easier to operate than the current BAM method for detection of Salmonella in cloves.

Survey of Foodborne Pathogens, Aerobic Plate Counts, Total Coliform Counts, and Escherichia coli Counts in Leafy Greens, Sprouts, and Melons Marketed in the United States

The objective of this research was to assess the microbiological status of leafy greens, sprouts, and melons from U.S. markets. A total of 14,183 samples of leafy greens, 2,652 samples of sprouts, and 3,411 samples of melons were collected throughout the United States from 2009 to 2014. The samples were analyzed for aerobic plate counts, total coliform counts, Escherichia coli counts, and the presence and levels of Salmonella, Shigella, Listeria monocytogenes, and Shiga toxin-producing E. coli (STEC), depending on the year and type of produce. Among the leafy greens, no E. coli O157:H7 or non-O157 STEC were detected from iceberg lettuce samples. The overall prevalences of Salmonella, E. coli O157:H7, non-O157 STEC, and L. monocytogenes in the 14,183 samples of leafy greens were 0.05, 0.01, 0.07, and 0.11%, respectively. Among sprout samples, no Salmonella or E. coli O157:H7 was detected, and the overall prevalences of non-O157 STEC and L. monocytogenes were 0.04 and 0.11%, respectively. Among melon samples, no Salmonella was detected from cucumbers, no L. monocytogenes was detected from cantaloupes, and the overall prevalences of Salmonella and L. monocytogenes were 0.12 and 0.23%, respectively. L. monocytogenes levels were 0.4 to 1,470 most probable number (MPN)/g in leafy greens, 0.36 to 1,100 MPN/g in sprouts, and <0.03 to 150 MPN/g in melons, and most positive samples had low levels of these pathogens. The isolates from these foods were very diverse genetically. Foodborne pathogens, including Salmonella, STEC, and L. monocytogenes, had relatively low prevalences in the produce surveyed. Because these foods are usually consumed raw, measures should be taken to significantly minimize the presence and levels of human pathogens.

Prevalence of Salmonella in 11 Spices Offered for Sale from Retail Establishments and in Imported Shipments Offered for Entry to the United States

The U.S. Food and Drug Administration conducted a survey to evaluate Salmonella prevalence and aerobic plate counts in packaged (dried) spices offered for sale at retail establishments in the United States. The study included 7,250 retail samples of 11 spice types that were collected during November 2013 to September 2014 and October 2014 to March 2015. No Salmonella-positive samples (based on analysis of 125 g) were found among retail samples of cumin seed (whole or ground), sesame seed (whole, not roasted or toasted, and not black), and white pepper (ground or cracked), for prevalence estimates of 0.00% with 95% Clopper and Pearsons confidence intervals of 0.00 to 0.67%, 0.00 to 0.70%, and 0.00 to 0.63%, respectively. Salmonella prevalence estimates (confidence intervals) for the other eight spice types were 0.19% (0.0048 to 1.1%) for basil leaf (whole, ground, crushed, or flakes), 0.24% (0.049 to 0.69%) for black pepper (whole, ground, or cracked), 0.56% (0.11 to 1.6%) for coriander seed (ground), 0.19% (0.0049 to 1.1%) for curry powder (ground mixture of spices), 0.49% (0.10 to 1.4%) for dehydrated garlic (powder, granules, or flakes), 0.15% (0.0038 to 0.83%) for oregano leaf (whole, ground, crushed, or flakes), 0.25% (0.03 to 0.88%) for paprika (ground or cracked), and 0.64% (0.17 to 1.6%) for red pepper (hot red pepper, e.g., chili, cayenne; ground, cracked, crushed, or flakes). Salmonella isolates were serotyped, and genomes were sequenced. Samples of these same 11 spice types were also examined from shipments of imported spices offered for entry to the United States from 1 October 2011 to 30 September 2015. Salmonella prevalence estimates (based on analysis of two 375-g composite samples) for shipments of imported spices were 1.7 to 18%. The Salmonella prevalence estimates for spices offered for sale at retail establishments for all of the spice types except dehydrated garlic and basil were significantly lower than estimates for shipments of imported spice offered for entry.