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THE INCIDENCE AND PERSISTENCE of Listeria monocytogenes
IS ASSOCIATED WITH ENVIRONMENT MICROBIOTA IN TREE FRUIT PACKING FACILITIES
Center for Food Safety and Applied Nutrition, U.S. Food and Drug AdministrationCollege Park, MD, U.S.
Dumitru Macarisin
www.fda.gov
AN INCREASE IN THE IMPLICATION OF TREE FRUITS IN OUTBREAKS AND RECALLS DUE TO CONTAMINATION BY FOODBORNE PATHOGENS
2011 Multistate Outbreak of Salmonella Agona Infections Linked to Imported Papayas (106 cases)
2012 Multistate Outbreak of Salmonella Braenderup Infections Linked to Mangoes (127 cases)
2012 Outbreak of Salmonella Illness Linked to Mangoes in Canada (21 cases)
2014 Outbreak of Listeriosis Linked to Stone Fruit in the USA (2 cases)
2015 Multistate Outbreak of listeriosis linked to prepackaged caramel apples (35 cases)
2017 Multistate Outbreak of Salmonella Urbana Infections Linked to Maradol Papayas (7 cases)
2017 Outbreak of Listeriosis Linked to Caramel Apples (3 cases)
2017 Multistate Outbreak of Salmonella Newport and S. infantis Infections Linked to MaradolPapayas (4 cases)
2017 Multistate Outbreak of Salmonella Anatum Infections Linked to Maradol Papayas (20 cases)
2019 Recall of whole avocados due to L. monocytogenes contamination
2019 Multistate Recall of Imported Stone Fruits Due to L. monocytogenes contamination
2019 Multistate Outbreak of Salmonella Infections Linked to Cavi Brand Papayas (71 cases)
SOURCES AND ROUTES OF TREE FRUIT CONTAMINATION
Ò Demonstrated: Ò Suspected:Ò Unknown:
Knowledge gap~5%~ 95 %
Bordini et al. Food Control 18 (2007) 1002–1007.
2014-2015 CARAMEL APPLE OUTBREAK INVESTIGATION
Ø Whole apples used in preparation of caramel apples were the ingredient contaminated by L. monocytogenes
Ø All whole apples were contaminated at a single packing facility in California
WHY L. monocytogenes?
PathogenEstimated annual # illnesses
Estimated annual # of deaths
Listeria monocytogenes 1,600 255 (16%
of cases)
Salmonella 1.0 million 378 (0.03% of cases)
Norovirus 5.5 million 149 (0.002% of cases)
FEW CASES, SEVERE
EXTREME SURVIVOR IN FOOD AND FOOD PRODUCTION ENVIRONMENTS
• Grows at -1.5°C and 45°C
• Higher virulence of cultures grown at 4°C comparing to those grown at 37°C
• Extreme halotolerance, survives over one year in 23.8% NaCl
• Superior barotolerance,survives over 150s at 400 MPa
APPROACH AND PREVALENT KNOWLEDGE GAPS
Ø Longitudinal surveillance of apple and stone fruit production environments for L. monocytogenes occurrence
v Pre-harvestv Post-harvest
• Seasonal variations• Processing/packing plants• Areas of a plant
Ø Whole genome sequencing of all L. monocytogenes isolatesv Persistent genotypesv Characterize inter-zone transfer in fruit packing facilitiesv Identify the development of AR in L. monocytogenes
Ø Microbiome analysis of post-harvest environmental samples Ø Evaluate the role/risk of convectional postharvest practices on L. monocytogenes
survival/transmission in apple production continuum
MICROBIOME STUDY DESIGN
Ø Three Apple Fruit Packing Facilities
Ø 13 standardized sample location (non-food contact surfaces)
Ø 3 sampling time points
PROCESSING AREA
SEASONAL DIFFERENCES IN L. monocytogenes OCCURRENCE
A significant (p < 0.05) association of season and L. monocytogenes incidence in facilities
Ò FallØ Increased volume of apples due to local harvestØ Increased organics and microbial load introduced into
the apple packing facilities Ò Winter
Ø Moderate volumes of apples processed from CA storageØ Due to lower temperatures, less competition with
background microbiota for L. monocytogenes
Ò SpringØ Decreasing volumes of apples packed due to lack of supplyØ Decreasing availability of organic and moisture due to
non-operation status
SEASONAL DIFFERENCES:Confirmed L. monocytogenes samples by season
L. monocytogenes positive samples
20 30 40 50 60 70
Fall
Winter
Spring
Year 1Year 2
4348
6442
3222
WHOLE GENOME SEQUENCING AND SEQUENCE ANALYSIS
Ø APPROACHÒ WGS was performed using a MiSeq (Illumina, Inc.) with the version 2 kit (2_250 bp)
Ò Core genome multi locus sequence type (cgMLST) was performed to analyze the genotypic diversity of the isolates
Ò Strain: cgMLST cluster + 12 allele difference
Ø DEFINING THE CRITERIA AND DETERMINING THE NUMBER OF ISOLATES Ò Isolates of the same strain from the same sample are counted as 1 unique isolate
Ò Isolates of the same strain from different samples are all counted
Ò Year 1 (2016-2017) generated 616 isolates
SUMMARY OF THE PRESENCE (+) AND ABSENCE (-) OF L. monocytogenes IN ENVIRONMENTAL SAMPLES COLLECTED FROM 3 APPLE PACKING FACILITIES
Chi-square test of L. monocytogenes occurrence among processing sections
L. monocytogenes occurrence Sectiona
Dry Wash Wax Absent 18 12 21 Present 21 27 18
Chi-Square DF P-value Pearson 4.38 2 0.112 Likelihood 4.454 2 0.108
aDF, degree of freedom.
Chi-square test of L. monocytogenes occurrence among facilities L. monocytogenes occurrence Facilitya
F1 F2 F3 Absent 28 0 23 Present 11 39 16
Chi-Square DF P-value Pearson 46.51 2 < 0.001 Likelihood 61.07 2 < 0.001
aDF, degree of freedom.
Adopted from: Tan, X., et al. (2019). Microbiome, 7(1), 1-18.
BACTERIAL ALPHA DIVERSITY DISTRIBUTIONS DETERMINED FOR SAMPLES COLLECTED FROM PACKING FACILITIES, F1, F2 AND F3.
aa
b
aa
b
Adopted from: Tan, X., et al. (2019). Microbiome, 7(1), 1-18.
MYCOBIOME ALPHA DIVERSITY DISTRIBUTIONS DETERMINED FOR SAMPLES COLLECTED FROM PACKING FACILITIES, F1, F2 AND F3.
a
ab
a
acab
Adopted from: Tan, X., et al. (2019). Microbiome, 7(1), 1-18.
CLUSTERING OF BACTERIAL COMMUNITIES BASED ON THE UniFracDISTANCES CALCULATED USING 16S rRNA V4 GENE SEQUENCES
Adopted from: Tan, X., et al. (2019). Microbiome, 7(1), 1-18.
CLUSTERING OF FUNGAL COMMUNITIES BASED ON THE UniFrac DISTANCES CALCULATED USING ITS2 SEQUENCES
Adopted from: Tan, X., et al. (2019). Microbiome, 7(1), 1-18.
RELATIVE ABUNDANCES OF BACTERIAL FAMILIES IN SAMPLES COLLECTEDIN THREE APPLE PACKING FACILITIES
In association with presence (+) or absence (-) of L. monocytogenes
In function of the location (washing, fan drying and waxing) within facilities
Adopted from: Tan, X., et al. (2019). Microbiome, 7(1), 1-18.
RELATIVE ABUNDANCES OF FUNGAL FAMILIES IN SAMPLES COLLECTEDIN THREE APPLE PACKING FACILITIES
In association with presence (+) or absence (-) of L. monocytogenes
In function of the location (washing, fan drying and waxing) within facilities
Adopted from: Tan, X., et al. (2019). Microbiome, 7(1), 1-18.
SUMMARY
Ø L. monocytogenes sporadically occurs in apple packing houses; however, in some facilities the contamination can become widespread and persistent.
Ø Higher bacterial and fungal diversity is associated with reduced occurrence of L. monocytogenes in packing houses. Whereas lower alpha diversity is associated with higher occurrence and persistence of L. monocytogenes in fruit packing facilities.
Ø Increased abundance of Pseudomonadaceae and Dipodascaceae organisms positively correlated with L. monocytogenes persistence in fruit processing environments.
Ø Current study provides baseline data needed for further in-depth investigation of microbial interactions between non-pathogenic and pathogenic microorganisms found in food processing environments.
Ø Further research in the area may lead to the optimization of pathogen control strategies and the development of novel, complementary, biocontrol methods to improve food safety.
HYPOTHESIS:
OBJECTIVES: Ò Determine if apple coating with wax can facilitate the survival of L. monocytogenes on
whole apples during long-term (< 5 months) storage
Ò Determine the effect of apple cultivar on the survival of L. monocytogenes in apple calyces and stem areas during prolonged cold storage
Ò Compare the survival of L. monocytogenes strains from the caramel apple outbreak, other outbreaks, tree fruit production environments and apples during prolonged cold storage
If apples become contaminated by L. monocytogenes during or after washing operations, wax coating may facilitate the survival of this pathogen on whole fruit during subsequent storage.
EXPERIMENTAL DESIGN
Ò Red Delicious, Granny Smith and Fuji apples were spot-inoculated in calix and stem areas with a six-strain cocktail of L. monocytogenes
Ò Half of inoculated fruits were subjected to coating with shellac wax
Ò L. monocytogenes populations in on waxed and un-waxed apples were enumerated in the course of prolonged storage at 4◦C
L. monocytogenes SURVIVAL ON APPLES AS AFFECTED BY THE APPLICATION OF WAX
LEGEND:Changes in L. monocytogenes populations
in waxed ( ) and un-
waxed ( ) apples were
assessed by direct plating
(LOD 1.39 log CFU/apple;
dotted line) and by the MPN
analysis ( in waxed
and in un-waxed
apples). Data represent the
means (n = 10) ± s.e.
Asterisks (black for direct
plating and red for MPN)
indicate values that are
statistically significantly
different (p < 0.05) in waxed
from corresponding values in
un-waxed apples.
Enumeration Method
Cultivar Storage time (days)
0 1 3 7 16 30 62 93 160
Direct plating,
log CFU/apple
Granny Smith a 3.85 (0.09) a 3.93 (0.10) a 3.74 (0.10) a 3.48 (0.08) a 3.06 (0.22) a 2.97 (0.27) a 2.79 (0.41)
Red Delicious a 3.99 (0.07) ab 3.74 (0.05) ab 3.61 (0.07) ab 3.20 (0.14) a 2.83 (0.21) b 2.53 (0.17) b 1.89 (0.15)
Fuji b 5.13 (0.06) ac 3.85 (0.04) ac 3.83 (0.04) ac 3.65 (0.05) b 3.51(0.03) a 3.20 (0.05) a 2.60 (0.13)
MPN, log
CFU/apple
Granny Smith 2.01 (0.53) a 1.22 (0.28) a 0.62 (0.50) a
Red Delicious 1.76 (0.13) ab 0.17 (0.18) b 0.41 (0.30) a
Fuji 2.54 (0.14) ac 0.97 (0.20) a 1.30 (0.42) a
MODERATE BUT SIGNIFICANT EFFECT OF CULTIVAR
Values represent the means (n = 10) and numbers in parenthesis indicate the standard error (s.e.). For direct plating data, values in the same column that are preceded by a different letter are significantly (p < 0.0167) different from each other. For MPN data, values in the same column that are followed by a different letter are significantly (p < 0.0167) different from each other.
Table 1. The effect of apple cultivar on Listeria monocytogenes survival on un-waxed apples
Enumeration Method
Cultivar Storage time (days)
0 1 3 7 16 30 62 93 160
Direct plating,
log CFU/apple
Granny Smith a 3.39 (0.09) a 2.62 (0.17) a 2.55 (0.07) a 2.48 (0.10) ab 1.90 (0.20) a 2.46 (0.31) a 2.36 (0.23)
Red Delicious b 2.94 (0.12) a 2.77 (0.11) a 2.53 (0.18) a 2.42 (0.19) ac 2.59 (0.15) a 1.95 (0.19) b 2.08 (0.21)
Fuji c 3.92 (0.15) b 1.83 (0.13) a 2.45 (0.27) a 2.47 (0.22) a 2.20 (0.15) a 1.91 (0.23) a 2.33 (0.20)
MPN, log
CFU/apple
Granny Smith 2.32 (0.19) a 1.77 (0.34) a 2.38 (0.24) a
Red Delicious 2.32 (0.14) a 1.66 (0.22) ab 2.58 (0.17) a
Fuji 2.48 (0.17) a 2.42 (0.15) ac 2.43 (0.11) a
Values represent the means (n = 10) and numbers in parenthesis indicate the standard error (s.e.). For direct plating data, values in the same column that are preceded by a different letter are significantly (p < 0.0167) different from each other. For MPN data, values in the same column that are followed by a different letter are significantly (p < 0.0167) different from each other.
Table 2. The effect of apple cultivar on Listeria monocytogenes survival on waxed apples
MODERATE BUT SIGNIFICANT EFFECT OF CULTIVAR
REPORTS:
1SCIENTIFIC REPORTS | (2019) 9:12170 | https://doi.org/10.1038/s41598-019-48597-0
www.nature.com/scientificreports
Survival of outbreak, food, and environmental strains of Listeria monocytogenes on whole apples as affected by cultivar and wax coatingDumitru Macarisin1, Ishani Sheth1, Minji Hur1, Anna Wooten1, Hee Jin Kwon1, Zhujun Gao1, Antonio De Jesus1, Wayne Jurick II2 & Yi Chen1
The 2014–2015 U.S. nationwide outbreak of listeriosis linked to apples used in commercially produced, prepackaged caramel apples was the first implication of whole apples in outbreaks of foodborne illnesses. Two case patients of this outbreak didn’t consume caramel apples but did eat whole apples, suggesting that contaminated whole apple may serve as a vehicle for foodborne listeriosis. The current study evaluated the effect of conventional fruit coating with wax and that of apple cultivar on the survival of outbreak-associated and non-outbreak Listeria monocytogenes strains on Red Delicious, Granny Smith and Fuji apples during 160 days under simulated commercial storage. L. monocytogenes survived in calyxes and stem ends of apples of all 3 cultivars through the duration of the experiment. After 2 months of storage, significantly (p < 0.05) larger L. monocytogenes populations were recovered from apples coated with wax than those un-waxed, regardless of the cultivar. No differences in survival amongst L. monocytogenes strains (serotypes 1/2a and 4b) from clinical, food, and environmental sources were observed. The observation that coating with wax facilitates prolonged survival of L. monocytogenes on whole apples is novel and reveals gaps in understanding of microbiological risks associated with postharvest practices of tree fruit production.
To date, two multistate outbreaks of Listeria monocytogenes infections were associated with the consumption of caramel apples. In the recent U.S. multistate listeriosis outbreak in 2017, three illnesses were caused by indis-tinguishable L. monocytogenes strains and the epidemiological investigation revealed that caramel apples were the likely source of the outbreak1. The earlier multinational outbreak of listeriosis in the U.S. and Canada in 2014–2015 was linked to whole apples used in commercially produced caramel apples and resulted in 35 illnesses and 7 deaths2. Thirty-one of the outbreak case patients reported eating commercially produced, prepackaged caramel apples before becoming ill2. The follow-up investigation determined that all implicated caramel apples were prepared using fruits from a single grower (Producer A) and demonstrated that fresh whole apples were the contaminated ingredient3. L. monocytogenes was also isolated from whole apples distributed in retail by Producer A and collected at various points in the distribution chain3.
Following the 2014–2015 caramel apple outbreak much of the research efforts were focused on understanding how L. monocytogenes in caramel apples could reach levels that were of significant risk to humans4,5. However, 3 of the case patients of this outbreak did not consume caramel apples, and actually ate fresh cut (one patient) or whole (2 patients) apples3, which suggested that L. monocytogenes from whole apples could cause human illnesses. On the other hand, whole apples were reported not to support L. monocytogenes proliferation under room tem-perature storage (up to 2 weeks) and refrigerated storage (up to 3 months)5,6. The shelf life of whole apples is very long compared to other ready-to-eat fresh produce and fresh apples are preserved up to 12 months without
1Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, USA. 2Food Quality Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD, USA. Correspondence and requests for materials should be addressed to D.M. (email: [email protected])
Received: 9 January 2019Accepted: 8 August 2019Published: xx xx xxxx
OPEN
FDA-CFSAN, Division of
Microbiology:
Penn State University:
ACKNOWLEDGEMENTS
Ø Luke LaBorde, Ph.D. Department of Food Science
Ø Tobin Simonetti
Department of Food ScienceØ Jasna Kovac, Ph.D.
Department of Food ScienceØ Xiaoqing Tan,
Department of Food ScienceØ Kari Peter, Ph.D.
Department of Plant Pathology
Ø Yi Chen, Ph.D.Ø Ms. Quing Jin
Ø Genome Trakr Team
Ø Eric W. Brown, Ph.D.
USDA-ARS:
Ò Michael Wisniewski, Ph.D.
Appalachian Fruit Research Station, Kearneysville, WV
Ò Wayne Jurick, Ph.D.Food Quality LaboratoryBeltsville, Maryland