the incidence and persistence of listeria monocytogenes · 2019-10-02 · the incidence and...

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


MYCOBIOME ALPHA DIVERSITY DISTRIBUTIONS DETERMINED FOR SAMPLES COLLECTED FROM PACKING FACILITIES, F1, F2 AND F3.

a

ab

a

acab


CLUSTERING OF BACTERIAL COMMUNITIES BASED ON THE UniFracDISTANCES CALCULATED USING 16S rRNA V4 GENE SEQUENCES


CLUSTERING OF FUNGAL COMMUNITIES BASED ON THE UniFrac DISTANCES CALCULATED USING ITS2 SEQUENCES


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


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


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

the incidence and persistence of listeria monocytogenes · 2019-10-02 · the incidence and...

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