International Journal of Food Microbiology 369 (2022) 109614

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Effect of storage temperature and time on the behavior of Salmonella,

Listeria monocytogenes, and background microbiota on whole fresh avocados
(Persea americana var Hass)
Elisa Cabrera-Díaz a, Liliana Martínez-Chávez b, Porfirio Gutiérrez-González b,
Julia A. Pérez-Montaño b, Ma. Ofelia Rodríguez-García b, Nanci E. Martínez-Gonzáles b, *
a
Departamento de Salud Pública, CUCBA, Universidad de Guadalajara, Zapopan, Jalisco 45110, Mexico
b
Departamentos de Farmacobiología y Matemáticas, CUCEI, Universidad de Guadalajara, Guadalajara, Jalisco 44430, Mexico

A R T I C L E I N F O A B S T R A C T

Keywords: Avocados are popular fruits; however, contamination of whole fresh avocados and avocado products with
Bacterial pathogens foodborne pathogens has raised concern about their safety. Recalls and import alerts of avocado products due to
indicator microorganisms contamination with Listeria monocytogenes cause important economic losses. The behavior of Salmonella,
Refrigeration
L. monocytogenes, and background microbiota on whole fresh avocados at 5 and 25 ◦ C as affected by temperature
Room temperature
and time of storage was investigated. Whole fresh avocados were inoculated by immersion in suspensions
Rate of decline
Produce containing six rifampicin-resistant strains of Salmonella or L. monocytogenes, and stored at 5 ◦ C for 48 d, or at
25 ◦ C for 11 d. At selected sampling times, avocados were removed from storage and pathogens enumerated. The
log counts of both pathogens at each temperature were fitted to the Weibull distribution nonlinear model to
estimate kinetic parameters including the time for the first 1-log reduction (δ), the shape of the curve (ρ), and the
time for two (2-D) and three (3-D) log reductions. Salmonella and L. monocytogenes initial populations (approx. 7
log CFU/avocado) decreased during storage at 5 and 25 ◦ C; L. monocytogenes mean counts were higher than those
observed for Salmonella (P < 0.05). L. monocytogenes showed a lower rate of decline at 5 ◦ C when compared to
Salmonella. In general, the ability of both pathogens to survive on the surface of avocados stored at room tem­
perature was similar. Salmonella and L. monocytogenes counts decline over time on the epicarp of whole avocados;
however, if the initial number of cells is large enough, the pathogens could be present for large periods of time.
Simultaneously, psychrotrophic microorganisms (PM), aerobic plate count (APC), coliforms (C) and yeasts/
molds (Y/M) were enumerated from non-inoculated avocados stored at 5 and 25 ◦ C. Initial mean counts for PM,
APC, C and Y/M ranged from 6.1 to 6.6 log CFU/avocado and showed no change (P > 0.05) during storage at
both temperatures. Good agricultural and handling practices from farm to fork are crucial to prevent or minimize
contamination of whole avocados; otherwise, if large numbers of pathogens contaminate the fruit, they could
survive and be transferred to the pulp, or to other ready to eat foods, representing a risk for consumers.

1. Introduction to its nutritional benefits. Avocados contain important components for a

healthy diet, such as unsaturated fatty acids, dietary fiber and phyto­
Avocados are tropical fruits widely consumed worldwide and have chemicals; preliminary clinical trials indicate that avocado consumption
become popular fruits in recent years. The per capita consumption of helps to support cardiovascular health and may support weight man­
avocados increased from 0.9 kg in 2001 to nearly 3.6 kg in 2018 in the agement and healthy aging (Dreher and Davenport, 2013).
United States (US) (Statista, 2020b). In Mexico, the principal global As the production and trade of avocados increase, illness cases and
avocado producer, the per capita consumption was reported in about 8 outbreaks related to its consumption could be expected. From 1997 to
kg in 2017 (Statista, 2020a). This high intake is related to an increase in 2017, at least 69 outbreaks were associated to consumption of guaca­
production and availability, and to a higher demand by consumers due mole and dishes with multiple ingredients -that included avocados-

* Corresponding author at: Av. Marcelino García Barragán 1421, Guadalajara, Jalisco 44430, Mexico.
E-mail address: [email protected] (N.E. Martínez-Gonzáles).

https://doi.org/10.1016/j.ijfoodmicro.2022.109614
Received 2 February 2021; Received in revised form 30 December 2021; Accepted 4 March 2022
Available online 7 March 2022
0168-1605/© 2022 Published by Elsevier B.V.
E. Cabrera-Díaz et al. International Journal of Food Microbiology 369 (2022) 109614

contaminated with viruses, bacteria or parasites, causing 1738 illnesses whole Hass avocados at 5 ◦ C are available. The purpose of this study was
and 58 hospitalizations in the US (Centers for Disease Control and Pre­ to determine the effect of temperature and time of storage on the
vention [CDC], 2021). The most commonly identified etiological agents behavior of Salmonella, L. monocytogenes, and background microbiota on
were Hepatovirus A and Norovirus (n = 26), followed by Salmonella (n = the epicarp of whole fresh avocados.
12), pathogenic Escherichia coli (n = 4), and other bacterial pathogens (n
= 9). Although only one of those outbreaks was related to the con­ 2. Materials and methods
sumption of avocados contaminated with Listeria monocytogenes as re­
ported in the National Outbreak Reporting System (NORS) database 2.1. Selection of fruits
(CDC, 2021), several food safety recalls and import alerts for avocado
products contaminated with this pathogen have been issued (Food and A total of 560 avocados (Persea americana var. Hass) were purchased
Drug Administration [FDA], 2019a, 2019b, 2020); they cause a negative from a produce distribution center located in Guadalajara City, Mexico.
economic impact for the avocado industry. In 1993, the FDA released All fruits were unripe, unwaxed, unwashed, free of visual defects such as
the Import Alert #21-12 for refrigerated or frozen guacamole and pro­ bruises, cuts or abrasions, and weighing 200 ± 50 g. Unwaxed and un­
cessed avocado products from Mexico; this Import Alert is still in effect washed avocados were selected because washing and waxing of this fruit
and has included 40 companies whose products are subjected to before entering the national market or before they are exported is not a
detention without physical examination (FDA, 2020). Between 2008 general practice in Mexico; also because some packing facilities apply
and 2011, at least 14 companies located in Michoacán State, the main fungicidal compounds as part of the waxing process which could inter­
avocado producing area of Mexico, closed their facilities due to fere with our experiments. The fruits were transported to the laboratory
contamination problems with L. monocytogenes (Rodríguez-García, in less than 1 h and kept at room temperature (25 ± 5 ◦ C) overnight
2011b). before experiments; no washing or decontamination treatments were
Contamination of avocados with pathogenic bacteria may take place applied before inoculation.
at any step of their production chain. Salmonella and L. monocytogenes
were isolated from the surface of Hass avocados collected at farms 2.2. Bacterial strains
(García-Sánchez, 2008; Rodríguez-García, 2011a); the same Salmonella
serotypes were recovered from avocados and environmental samples The bacterial isolates used in this study were five Salmonella strains
including irrigation water, soil, manure and workers' shoes. In some kindly provided by Dr. Alejandro Castillo from Texas A&M University,
cases, Salmonella strains with indistinguishable PFGE profiles were identified as S. Poona (73S), S. Montevideo (2S), S. Michigan (12S), S.
recovered from avocados and soil samples (Rodríguez-García, 2011a). Gaminara (27S) and S. Agona (30S), that were originally isolated from
Further down in the supply chain, Salmonella and L. monocytogenes were feces of patients linked to produce outbreaks; and one strain of S.
recovered from domestic and imported avocados before they reached Newport (Sm-R-027) which was isolated by our research group from
retailing markets in the US (FDA, 2018a), and both pathogens were also produce irrigation water. Six L. monocytogenes isolates were also used
isolated from whole fresh avocados collected at retail markets in Mexico and included the strains California and Scott A ATCC 15313 (both of
(García-Frutos et al., 2020). To prevent or minimize the contamination serotype 4b and related to foodborne outbreaks), one strain recovered
of avocados with foodborne pathogens, it is crucial to comply with Good from broccoli (N-28) and two strains from guacamole (N-4 and N-5)
Agricultural Practices (GAP) and Good Handling Practices (GHP) kindly provided by Dr. Montserrat Iturriaga from the Autonomous
throughout their production chain. University of Querétaro, Mexico; and one strain (Lm-R-054) isolated
The avocado production process involves harvesting of the fruits at from avocado by our research group. Rifampin-resistant (Rif+) mutants
farms, transportation to packing facilities, tempering, either dry brush­ of each strain were obtained in our laboratory using the procedure
ing or washing plus sanitizing, and sometimes, waxing. The application described by Kaspar and Tamplin (1993) to facilitate their recovery and
of wax emulsions helps to reducing moisture loss, delay softening, and enumeration from the inoculated avocados. Stock cultures of each strain
improve appearance; however, this is not a general practice in the av­ were stored in tryptic soy broth with 20% glycerol at − 80 ◦ C for long-
ocado industry in Mexico. Afterwards, avocados are packed and stored term preservation. Working cultures were maintained at 5 ◦ C on
at temperatures between 3 and 7 ◦ C before distribution. The distribution tryptic soy agar (TSA) (Bioxon, Becton Dickinson, Mexico) for Salmo­
of avocados to wholesalers is conducted in refrigerated units to delay the nella, and on TSA supplemented with 0.6% yeast extract (TSAYE, Bio­
ripening process and later, during retailing at supermarkets, avocados xon) for L. monocytogenes, and sub-cultured every two months by
are commonly stored in cold chambers (<7 ◦ C) before displaying at transferring a loop to fresh TSA or TSAYE slants for Salmonella or
room temperature (≈25 ◦ C). In small local markets, the fruits are L. monocytogenes, respectively, with incubation at 35 ◦ C overnight. The
commonly stored and displayed at room temperature (García-Frutos purity and identity of all Salmonella cultures was confirmed in xylose
et al., 2020). At home, consumers keep avocados in the refrigerator to lysine deoxycholate agar (XLD, Bioxon) followed by biochemical testing
delay ripening or at room temperature until they reach the desired in triple sugar iron (TSI, Bioxon), lysin iron agar (LIA, Bioxon) and urea
ripeness. Storage of the fruit at room temperature accelerates its broth (Difco, Becton Dickinson, USA). For L. monocytogenes cultures,
ripening and may affect the survival or growth of mesophilic pathogens purity and identity was confirmed in Modified Oxford Agar (MOX,
and spoilage microorganisms. Rezende et al. (2016) reported an increase Difco) with further testing for Gram stain, fermentation of rhamnose,
in Salmonella counts on the epicarp and in the pulp of avocados (Persea xylose, and mannitol, and CAMP (Christie, Atkins, Munch-Petersen)
americana var. Americana) stored at 20 and 30 ◦ C, while Gómez-Aldapa assay. In addition, resistance to rifampicin was verified by streaking
et al. (2016) found that Staphylococcus aureus, Shigella flexneri, Salmo­ the cultures on TSA or TSAYE supplemented with rifampicin (100 μg/
nella Montevideo, S. Typhi, Vibrio cholerae and different pathotypes of mL, Sigma-Aldrich, St. Louis, USA) before the experiments.
E. coli were recovered from whole fresh avocados for at least 20 days at
22 ◦ C and 80% of relative humidity. 2.3. Inoculum preparation
Storage at low temperatures (<7 ◦ C) contributes to maintain
avocados freshness for a longer time by reducing respiration, enzymatic Mixtures containing the six described strains of Salmonella or the six
activity, ethylene production, water and firmness loss, and by slowing or strains of L. monocytogenes were prepared to inoculate the surface of
inhibiting spoilage microorganism's growth. Optimum storage temper­ avocados. To prepare the mixtures, each Salmonella strain was individ­
atures for Hass avocados are 5–7 ◦ C for early season fruit and 4–5.5 ◦ C ually cultured in 3 mL of TSB and each L. monocytogenes strain in 3 mL of
for late season fruit (Yahia, 2012). To the best of our knowledge, no TSBYE; all cultures were incubated at 35 ◦ C overnight. After incubation,
studies related to the behavior of Salmonella and L. monocytogenes on an aliquot of 0.5 mL from each culture was individually transferred to a

2
E. Cabrera-Díaz et al. International Journal of Food Microbiology 369 (2022) 109614

flask containing 70 mL of fresh TSB or TSBYE, and incubated at 35 ◦ C for decimal dilutions were prepared from the rinse liquid using PW and
18–24 h. The cultures were then divided into two conic tubes each plated on TSA-Rif or TSAYE-Rif as corresponded for each pathogen; all
containing 35 mL of the strain culture, centrifuged at 4507 ×g for 10 min plates were incubated at 35 ± 2 ◦ C and colonies enumerated after 24–48
at 4 ◦ C (Durafuge 300R, Precision™, Ohio, USA); the supernatant was h. The remaining volume of the rinse liquid was incubated at 35 ± 2 ◦ C
discarded and each pellet was washed in 35 mL of sterile saline solution for 18 h and then streaked on TSA or TSAYE plates to detect Salmonella
(SS, 0.85% NaCl), centrifuged at similar conditions and the pellets and L. monocytogenes in those samples showing counts below the
resuspended again in 35 mL of SS. For each pathogen, equal volumes detection limit of the spread plating technique. Several colonies from
(70 mL) were mixed in a sterile flask to obtain 420 mL of the washed each sample were confirmed by further testing including Gram stain and
cells mixture which was then diluted with 1580 mL of 0.1% peptone biochemical reactions on triple sugar iron agar, urea agar, and lysine
water (PW) at room temperature (25 ± 5 ◦ C) to obtain a final volume of iron agar for Salmonella (Andrews et al., 2016), and Gram stain,
2 L of washed cells inoculum that was transferred into a sterile Nal­ fermentation of rhamnose, xylose, and mannitol, and CAMP (Christie,
gene™ bucket. The bacterial concentration in each pathogen mixture Atkins, Munch-Petersen) test for L. monocytogenes (Ryser and Donnelly,
was determined by plating appropriate serial decimal dilutions prepared 2001).
in PW, on TSA or TSAYE supplemented with 100 μg of rifampicin/mL To enumerate microbial indicator groups on non-inoculated
(TSA-Rif, TSAYE-Rif); the colonies were enumerated after incubation at avocados sampled at the selected times after storage at 5 and 25 ◦ C,
35 ◦ C for 24 h. Initial counts of each inoculum mixture were ~7 log each fruit was placed in a sterile Whirl-Pak bag with 100 mL of PW and
CFU/mL. hand-massaged for 2 min. Serial decimal dilutions in PW were prepared
from the rinse and pour plated on plate count agar (Bioxon) for
2.4. Inoculation of avocados enumeration of PM and APC after incubation at 7 ◦ C ± 0.5 ◦ C for 10
d and at 35 ± 2 ◦ C for 48 h, respectively; on red violet bile agar (Bioxon)
Whole fresh avocados were surface inoculated with the Salmonella or with overlay for enumeration of coliforms at 35 ◦ C ± 2 ◦ C for 24 h; and
L. monocytogenes mixture to study the behavior of both pathogens under on potato dextrose agar (Bioxon) supplemented with rose bengal (200
two different storage temperatures. Groups of three avocados were μg/mL, Hycel, Mexico) and ampicillin (100 μg/mL, Sigma-Aldrich) for
immersed at a time in the Nalgene™ bucket containing the Salmonella or enumeration of Y/M at 25 ± 2 ◦ C for 3–5 d.
L. monocytogenes inoculum mixture at room temperature (25 ± 5 ◦ C);
each avocado was rotated for 1 min using a sterile glass rod to assure an
even inoculation of the surface. The inoculated avocados were trans­ 2.7. Data analysis
ferred to sterile buckets and let to drain for 15 min at room temperature,
then placed at a distance of 2 cm from each other into sanitized poly­ Prior to data analysis, microbial counts for Salmonella,
ethylene racks (50 × 30 × 6 cm). Immediately after, Salmonella and L. monocytogenes and microbial indicators were calculated as CFU per
L. monocytogenes were enumerated from two inoculated avocados. Each avocado, converted into log10 values (log) and mean values were
avocado was individually placed in a sterile Whirl-Pak bag (Nasco, calculated. One-way analysis of variance (ANOVA) was used to examine
Wisconsin, USA), added with 100 mL of sterile TSB or TSBYE and then the effect of storage at each temperature on Salmonella, L. monocytogenes
hand-massaged for 2 min; pathogens were enumerated as further or indicators mean counts. For each storage temperature, a multifacto­
described in Section 2.6. The initial mean counts at day 0 were ~7 log rial ANOVA was performed to determine the effect of time in the log
CFU/avocado. counts of either pathogens or microbial indicators. When significant
differences (P < 0.05) were observed, the Tukey's test was performed. To
describe the behavior of pathogens, the log counts for each temperature
2.5. Behavior of pathogens and microbial indicator groups at two
were fitted to the Weibull distribution nonlinear model according to Eq.
temperatures
(1) (Mafart et al., 2002):
Avocados inoculated with Salmonella or L. monocytogenes were stored ( t )ρ
log(N) = logN0 − (1)
under refrigeration conditions (5 ± 1 ◦ C) or at room temperature (25 ± δ
5 ◦ C). Temperature was monitored inside incubators using mercury
thermometers (1221G96 Accu-Lab, Thomas Scientific, NJ, USA) inser­ where N0 is the initial count of each pathogen, t is the time, δ is the time
ted into glass bottles filled with water and sealed. In addition, non- for the first log reduction on the pathogen count, and ρ is the shape of the
inoculated avocados were also stored at each temperature and used curve. The time to observe 2-log (2-D) and 3-log (3-D) reductions were
for enumeration of microbial indicator groups. Two inoculated avocados calculated by using the shape and scale parameters according to Eq. (2)
stored at 5 ◦ C were sampled to enumerate Salmonella or L. monocytogenes (Huang et al., 2013).
every 3 d and up to 48 d; while two inoculated avocados stored at 25 ◦ C txD = δ × (x)1/ρ (2)
were sampled at 4, 8, 12, 24, 28, 32, 36 and 48 h and then every 24 h for
a total of 264 h (11 d) and pathogen's counts determined. At the same One-way ANOVA was used to examine the effect of storage at each
sampling times, two non-inoculated avocados were removed from temperature on the rate of decline observed for Salmonella and
storage at 5 ◦ C to enumerate psychrotrophic microorganisms (PM) L. monocytogenes. The Statgraphics Centurion XV software (version
(Cousin et al., 2001), coliforms (C) (Feng et al., 2020) and yeasts and 15.2.06, Statpoint Technologies, Inc., Warrenton, VA, USA) was used.
molds (Y/M) (Beuchat and Cousin, 2001). In addition, two non-
inoculated avocados were removed from storage at 25 ◦ C to 3. Results and discussion
enumerate aerobic plate count (APC) (Maturin and Peeler, 2001), C and
Y/M. Two independent experiments were conducted. 3.1. Effect of storage on the behavior of pathogens on avocados stored at
two temperatures
2.6. Enumeration of pathogens and microbial indicator groups
Avocados are fruits exposed to numerous contamination sources
Salmonella and L. monocytogenes were enumerated from inoculated from farm to home environments; these may include irrigation water,
avocados at selected times after storage at 5 and 25 ◦ C. Each avocado soil, compost, wild and domestic animals, harvesting utensils, equip­
was individually placed in a sterile Whirl-Pak bag (Nasco, Wisconsin, ment and water in post-harvest operations, workers, transports, and
USA), added with 100 mL of sterile TSB or TSBYE and then hand- diverse contact surfaces. Through these sources, pathogens like Salmo­
massaged for 2 min. The fruit was removed from the bag and serial nella and L. monocytogenes may reach the surface of whole avocados,

3
E. Cabrera-Díaz et al. International Journal of Food Microbiology 369 (2022) 109614

where several intrinsic and extrinsic factors of the fruit may influence (Wang et al., 2009). It is possible that the inoculation method used also
their growth or survival. contributed to this variation observed; when whole avocados were
The group of avocados that were inoculated and stored at 5 ± 1 ◦ C immersed into the inoculum mixture, a larger uneven rough surface area
showed an initial mean concentration of Salmonella of 7.4 log CFU/av­ was inoculated resulting in microbial counts with larger variation.
ocado at day 0 which significantly decreased (P < 0.05) to 4.4 log CFU/ The kinetic parameter estimates based on the Weibull distribution
avocado after 3 d under cold storage (Table 1) and continued declining model (Table 2) indicate that Salmonella and L. monocytogenes pop­
over time. After 45 d of storage, mean counts were below the detection ulations declined at 5 and 25 ◦ C at different rates. The fitted curves
limit (2.0 log CFU/avocado); however, the pathogen was still recovered showed a concave shape (ρ < 1) demonstrating a gradual reduction over
from the TSB used to rinse the epicarp, after incubation at 35 ± 2 ◦ C for time (Figs. 1 and 2). At 5 ◦ C, the mean time for the first 1-log reduction
18 h. For those avocados inoculated with L. monocytogenes, the initial (δ) was 0.060 d (1.4 h) for Salmonella and 0.08 d (1.9 h) for L. mono­
mean concentration on the surface of avocados stored at 5 ± 1 ◦ C was cytogenes, and the mean time for a 2-log reduction (2-D) was 0.90 d (22
7.4 log CFU/avocado at day 0, and likewise Salmonella populations, h) and 1.5 d (36 h), respectively; these rates of decline were not different
significantly decreased to 5.5 log CFU/avocado after 3 d of cold storage for both pathogens (P > 0.05). However, the time for a 3-log reduction
(P < 0.05). Although L. monocytogenes populations continued decreasing (3-D) was longer for L. monocytogenes (P < 0.05) indicating a lower rate
over time, mean counts of 3.2 log CFU/avocado were observed after 45 of decline and a better ability to survive at 5 ◦ C when compared to
d of cold storage. Despite its psychrotrophic nature, L. monocytogenes Salmonella. At 25 ◦ C, the mean δ values for Salmonella and
was not able to grow on the epicarp of avocados stored at 5 ◦ C. The L. monocytogenes were 0.14 and 2.6 h, respectively, and showed no
prolonged cold storage produced a significant reduction on both path­
ogens' populations (P < 0.05); however, L. monocytogenes mean counts
Table 2
were significantly higher than those observed for Salmonella (P < 0.05).
Parameter estimates for Salmonella and Listeria monocytogenes on the surface of
On avocados inoculated with Salmonella and stored at 25 ◦ C, the avocados (Persea americana var. Hass) at 5 and 25 ◦ C based on the Weibull
initial mean counts were 7.2 log CFU/avocado and significantly survival model.
decreased to 5.3 log CFU/avocado (P < 0.05) after 8.0 h (0.3 d) of
Temperature Parameterb Mean values ± SDa
storage, then remained with no significant change for at least 264 h (11
d) of storage (Table 1). At the same temperature, L. monocytogenes Salmonella L. monocytogenes

populations significantly decreased from initial 7.5 log CFU/avocado at 5 ◦C δ (d) 0.06 ± 0.04 ac 0.08 ± 0.10 a
time 0, to 5.3 log CFU/avocado after 24 h (1 d) of storage (P < 0.05), and ρ 0.25 ± 0.03 0.19 ± 0.07
R2 0.76 ± 0.09 0.71 ± 0.11
then persisted with no significant changes until 264 h (11 d) of storage.
2-D 0.90 ± 0.35 a 1.5 ± 1.0 a
The prolonged storage also resulted in significant reductions for both 3-D 4.5 ± 1.1 a 13 ± 6.5 b
pathogens (P < 0.05), although L. monocytogenes generally showed 25 ◦ C δ (h) 0.14 ± 0.15 a 2.6 ± 2.1 a
higher mean counts than Salmonella (P < 0.05). ρ 0.14 ± 0.03 0.24 ± 0.06
A large variation on the pathogens counts was observed at both R2 0.55 ± 0.11 0.66 ± 0.07
2-D 16 ± 1.1 a 38 ± 16 b
temperatures over storage. The roughness of avocado epicarp differs 3-D 371 ± 182 a 214 ± 17 a
among fruit pieces and may account for this large variation. The re­
a
covery of the inoculated pathogens cells was probably less efficient from Mean value represent the mean of four replicates ± standard deviation.
b
δ represent the mean time for the first log reduction, ρ is the shape of the
those avocados with higher surface roughness. Previous studies have
curve, 2-D and 3-D are the time for two and three log reductions, respectively;
demonstrated that bacterial adhesion rate increase as the surface
time is given in days at 5 ◦ C and in hours at 25 ◦ C.
roughness of fruits raise, inducing protection for microorganisms c
Mean values within rows with different letters (a, b) are significantly
entrapped on the surface and resulting in reduced washing efficacy different (P < 0.05).

Table 1
Mean counts of Salmonella and Listeria monocytogenes on the surface of avocados (Persea americana var. Hass) as affected by storage time at 5 and 25 ◦ C.
Days Mean counts log CFU/avocado ± SDa

5 ◦C Hours 25 ◦ C

Salmonella L. monocytogenes Salmonella L. monocytogenes

b
0 7.4 ± 0.18 e 7.4 ± 0.15 c 0 7.2 ± 0.16 f 7.5 ± 0.11 e
3 4.4 ± 0.81 d 5.5 ± 0.46 b 4 5.8 ± 0.16 e 6.8 ± 0.37 de
6 3.7 ± 0.83 bcd 5.0 ± 0.58 ab 8 5.3 ± 0.43 cde 6.6 ± 0.45 cde
9 3.8 ± 0.22 bcd 4.6 ± 0.63 ab 12 5.3 ± 0.21 cde 6.1 ± 0.22 bcde
12 3.8 ± 1.1 bcd 3.6 ± 0.36 a 24 5.7 ± 1.1 de 5.3 ± 0.23 abc
15 4.2 ± 1.7 cd 3.8 ± 0.60 a 28 4.6 ± 0.81 abc 5.4 ± 0.58 abcd
18 2.7 ± 0.25 abcd 3.9 ± 0.90 ab 32 4.9 ± 0.56 bcde 5.6 ± 0.33 abcd
21 2.5 ± 0.91 abc 4.6 ± 0.19 ab 36 4.9 ± 0.46 bcde 5.4 ± 0.82 abcd
24 2.4 ± 0.47 abc 3.8 ± 0.28 a 48 4.6 ± 0.50 abc 4.9 ± 0.19 ab
27 3.7 ± 0.32 bcd 3.9 ± 0.65 ab 72 4.9 ± 0.47 bcde 5.2 ± 0.91 abc
30 2.4 ± 0.55 abc 3.7 ± 0.71 a 96 4.9 ± 0.79 bcde 4.5 ± 0.95 a
33 2.5 ± 0.54 abc 4.2 ± 0.83 ab 120 4.2 ± 0.45 ab 4.7 ± 0.23 a
36 2.2 ± 0.93 ab 3.9 ± 0.92 ab 144 4.7 ± 0.78 abcd 4.3 ± 0.30 a
39 2.4 ± 0.47 abc 3.5 ± 0.27 a 168 4.6 ± 0.83 abc 4.4 ± 0.57 a
42 2.2 ± 0.65 ab 4.4 ± 0.77 ab 192 4.4 ± 1.1 abc 4.2 ± 0.80 a
45 1.7 ± 0.00 ac 3.7 ± 1.2 a 216 3.8 ± 1.1 a 4.5 ± 0.78 a
48 1.7 ± 0.00 a 3.2 ± 0.31 a 240 4.6 ± 0.95 abc 5.0 ± 0.79 ab
264 4.4 ± 0.61 abc 5.1 ± 0.23 ab
a
Counts represent the mean of four replicates ± standard deviation.
b
Mean values within columns with different letters (a, b, c, d, e) are significantly different (P < 0.05).
c
The minimum detection limit was 100 CFU/avocado (2.0 log CFU/avocado); because the pathogen was always detected after enrichment of the sample, an in­
termediate value between 1 and 99 CFU/avocado (1.7 log CFU/avocado) was used for the statistical analysis.

4
E. Cabrera-Díaz et al. International Journal of Food Microbiology 369 (2022) 109614

8 7.3
R1 R1

(LogCFU/avocado)
Salmonella (Log CFU/avocado)
6 6.3

Listeria(Log 10(N))
4 5.3

L. monocytogenes
2 4.3

0 3.3
0 10 20 30 40 50 0 10 20 30 40 50
Time Time

8 7.9
R2 R2

(LogCFU/avocado)
Salmonella (Log CFU/avocado)

6.9
6

10(N))
5.9

Listeria(Log
4

L. monocytogenes
4.9

2
3.9

0 2.9
0 10 20 30 40 50 0 10 20 30 40 50
Time Time

8 7.5
R3 R3
(LogCFU/avocado)
Salmonella (Log CFU/avocado)

6.5
6
10 (N))

5.5
Listeria(Log

4
L. monocytogenes

4.5

2
3.5

0 2.5
0 10 20 30 40 50 0 10 20 30 40 50
Time Time

8 7.8
R4
(LogCFU/avocado)

R4
Salmonella (Log CFU/avocado)

6.8
10(N))

5.8
Listeria(Log

4
L. monocytogenes

4.8

2
3.8

0 2.8
0 10 20 30 40 50 0 10 20 30 40 50
Time Time

Fig. 1. Survival curves of Salmonella and Listeria monocytogenes on whole fresh avocados stored at 5 ◦ C. Time is given in days. Two independent experiments were
conducted in duplicate and each curve represents a single replicate. Data were fitted to Weibull model.

significant differences (P > 0.05). The mean time for 2-D was signifi­ Enteritidis and S. Montevideo on the epicarp of avocados previously
cantly longer for L. monocytogenes (38 h) than for Salmonella (16 h) (P < sanitized with chlorine solution (150 mg/L). Portions of 2 cm2 (2 g) of
0.05); however, afterwards, the rate of decline was not different for both avocados epicarp were aseptically removed, stored at − 20 ◦ C, and
pathogens (P > 0.05). This indicates that in general, the ability of both defrosted before inoculation, then stored at 10, 15, 20 and 30 ◦ C. Ac­
pathogens to survive on the surface of avocados stored at room tem­ cording to this study, Salmonella was able to grow on the epicarp of
perature was similar. avocados at all temperatures showing higher growth rates as the tem­
Despite the reduction observed in Salmonella and L. monocytogenes perature increased. Contrary to the results reported by Rezende et al.
populations, both pathogens were recovered from the surface of whole (2016), we found that Salmonella was not able to grow on the epicarp of
fresh Hass avocados after prolonged storage at 5 ◦ C and 25 ◦ C. This could whole avocados stored at room temperature, yet the populations
have been influenced by the high concentration used to inoculate both decreased from 7.2 to 4.4 log CFU/avocado after 264 h (11 d). These
pathogens on the surface of avocados, which may not represent the most differences may be due to experimental designs used in both studies;
common situation, although exemplifies the worst-case scenario. inoculating small portions of the epicarp that were previously frozen and
Studies reporting the behavior of Salmonella and L. monocytogenes on thawed, as reported by Rezende et al. certainly do not represent the real
avocados are scarce, and the few available show important differences in conditions that microorganisms encounter when they are attached to the
the methodology used to inoculate the fruit surface; these differences epicarp of whole fresh avocados. Avocados are climacteric fruits with
may account for the dissimilar results observed among studies. Rezende high metabolic activity and large gas exchange during their ripeness
et al. (2016) inoculated a mixture of Salmonella Typhimurium, S. process after harvesting (Villa-Rodríguez et al., 2011) which create

5
E. Cabrera-Díaz et al. International Journal of Food Microbiology 369 (2022) 109614

og 1CFU/avocado)
7.8
R1 R1

ella (CFU/avocado)
6.8 7

Log10(N))

0(N))
6

Listeria (L(Log
5.8
Salmon(Log

L. monocytogenes
4.8 5
Salmonella

3.8 4

2.8 3
0 50 100 150 200 250 300 0 50 100 150 200 250 300
Time Time

8.1 7.6

og10CFU/avocado)
R2 R2
ella(LCFU/avocado)

7.1
og10(N))

6.6

Listeria (L(Log (N))

6.1
Salmon(Log

5.6

L. monocytogenes
5.1
Salmonella

4.6
4.1

3.1 3.6
0 50 100 150 200 250 300 0 50 100 150 200 250 300
Time Time

7.5 8
R3
Log1CFU/avocado)
R3
lla (LCFU/avocado)
og10(N))

6.5 7
Listeria((Log 0(N))
Salmone(Log

5.5 6
L. monocytogenes
Salmonella

4.5 5

3.5 4
0 50 100 150 200 250 300 0 50 100 150 200 250 300
Time Time

7.7 7.9
og10CFU/avocado)

R4
ella(LCFU/avocado)

R4
og10(N))

6.7 6.9
Listeria(L(Log (N))
Salmon(Log

5.7 5.9
L. monocytogenes
Salmonella

4.7 4.9

3.7 3.9
0 50 100 150 200 250 300 0 50 100 150 200 250 300
Time Time

Fig. 2. Survival curves of Salmonella and Listeria monocytogenes on whole fresh avocados stored at 25 ◦ C. Time is given in hours. Two independent experiments were
conducted in duplicate and each curve represents a single replicate. Data were fitted to Weibull model.

specific characteristics on the epicarp that were not replicated by the mg/L, pH 5.0) for 5 min and then rinsed with sterile tap water. The
experiments conducted by Rezende et al. where avocados were first initial concentration of the pathogens (~4.5 log CFU/0.5 cm2) signifi­
disinfected, and then portions of the epicarp were dissected, frozen and cantly decreased over time and after 20 days of storage was reported in
defrosted prior to inoculation. When fruits are frozen and thawed, the 1.0–1.7 log CFU/0.5 cm2. Comparably to the report by Gómez-Aldapa
degradation of cellulose, hemicellulose and pectins of cell walls reduce et al. (2016), we found that Salmonella and L. monocytogenes were not
cell adhesion resulting in textural changes and softening (Phothiset and able to grow on the surface of whole avocados; however, those authors
Charoenrein, 2013; Charoenrein and Owcharoen, 2016). It is possible found that mean populations for both pathogens were not significantly
that changes induced by freezing and thawing affected the interaction of different (P > 0.05) over storage. Disparities in the experimental design,
Salmonella with the avocado surface; a portion of the pathogen's popu­ including the disinfection procedure of whole avocados before inocu­
lation could have died or been injured through freezing and thawing, but lation, the use of one single strain of each pathogen, instead of a mixture
survivors probably found a more suitable environment to grow. of different strains, as well as the smaller surface area inoculated may
In other experiments conducted by Gómez-Aldapa et al. (2016), the account for differences in the results observed between the two studies.
behavior of several foodborne pathogens including Salmonella Typhi, S. The ripening stage of avocados at the moment of inoculation may also
Typhimurium and L. monocytogenes, on the surface of avocados stored influence the attachment and behavior of foodborne pathogens on the
for 20 d at 22 ± 2 ◦ C was investigated; each pathogen was individually epicarp; unfortunately, this characteristic was not reported in those
inoculated over an area of 0.5 cm in diameter on the surface of whole studies conducted either by Rezende et al. (2016) or Gómez-Aldapa et al.
fresh avocados previously disinfected with sodium hypochlorite (200 (2016). Despite the differences among reports, Salmonella and

6
E. Cabrera-Díaz et al. International Journal of Food Microbiology 369 (2022) 109614

L. monocytogenes counts decline over time on the epicarp of whole (García-Sánchez, 2008). Failure to adhere to GAP was observed in both
avocados; however, if the initial number of cells is large enough, the studies; common detected problems included presence of wild and do­
pathogens could be present for long periods of time. This emphasizes the mestic animals in the farms, unprotected water sources, application of
importance of implementing GAP and GHP to prevent or minimize the non-treated soil amendments, deficient cleaning and sanitizing proced­
contamination that may take place at any step of their production chain. ures for harvesting tools, and poor hygiene practices among workers.
The microbial quality of irrigation water and soil amendments, control Further in the production chain, the presence of Salmonella and
of wild and domestic animals, hygiene practices of workers and sani­ L. monocytogenes on whole fresh avocados has also been reported. In a
tation practices are essential to minimize the presence of foodborne study conducted in the US, the overall prevalence of Salmonella on whole
pathogens on fresh avocados. fresh avocados before they reached the retail markets was calculated in
In Mexico, the isolation of Salmonella and L. monocytogenes was 0.7% (n = 1615), while the prevalence of L. monocytogenes in 18% (n =
previously reported in 3% (4/144) and 0.7% (1/144), respectively, of 361) (FDA, 2018a). In Mexico, we reported a frequency of isolation of 4
Hass avocados collected at farms (Rodríguez-García, 2011a); Salmonella and 8% (n = 225) for Salmonella and L. monocytogenes, respectively, on
was also isolated from environmental samples showing a frequency of the epicarp of whole avocados sold at local retail markets (García-Frutos
4% (27/720) in soil, 9% (16/180) in water, and 3% (5/163) in shoes of et al., 2020).
workers, harvesting utensils and door handles. In the same study, As demonstrated by those studies, it is possible to expect the presence
L. monocytogenes was isolated from 2% (13/725) of soil, 5% (9/180) of of Salmonella and L. monocytogenes on the surface of whole fresh
water, and 4% (7/163) of surfaces samples. In another investigation also avocados from where they can be transferred to the avocado pulp
conducted in Mexico, Salmonella was isolated from 3% (2/75) of Hass through punctures, wounds and cuts (Sharma and Luo, 2011), by
avocado composite samples collected at two exporting farms; serotypes internalization via the steam scar (Chen et al., 2016), or by cross-
Poona, Bardo and Newport were isolated from avocados and also from contamination during slicing or manipulation when preparing guaca­
environmental samples including soil, irrigation water and manure mole or other ready to eat foods. Preventing contamination of the

5 °C
8

6
Log CFU/avocado

0
0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48
Days
Psychrotrophs Coliforms Yeasts and molds

25 °C
8

7
Log CFU/avocado

0
0 4 8 12 24 28 32 36 48 72 96 120 144 168 192 216 240 264
Hours

Aerobic plate count Coliforms Yeasts and molds

Fig. 3. Mean counts (Log CFU/avocado) of microbial indicator groups on whole fresh avocados stored at 5 ◦ C and 25 ◦ C.

7
E. Cabrera-Díaz et al. International Journal of Food Microbiology 369 (2022) 109614

avocado pulp is very important, since it was demonstrated that Salmo­ 4. Conclusions
nella and L. monocytogenes can grow on avocado pulp and guacamole
stored at 4–7 ◦ C and at 22 ◦ C (Arvizu-Medrano et al., 2001; Iturriaga High initial populations of Salmonella and L. monocytogenes on whole
et al., 2002). In order to reduce the risk, the FDA recommends that fresh Hass avocados showed a gradual reduction over time. Both path­
consumers wash avocados thoroughly under running water and scrub ogens were recovered after prolonged storage under refrigeration and at
them with a clean produce brush before preparing and/or eating; the room temperature; still L. monocytogenes demonstrated a lower rate of
fruits should be dried with a clean cloth or paper towel to further reduce decline under refrigeration when compared to Salmonella. Hence, results
bacteria that may be present (FDA, 2018b). from the present study contribute to understand the behavior of Sal­
monella and L. monocytogenes on whole fresh avocados and could be
3.2. Effect of storage on levels of background microbiota at two further used to conduct microbial risk assessments. Good agricultural
temperatures and handling practices from farm to fork are crucial to prevent or
minimize contamination of avocados; otherwise, surviving pathogens
The levels of background microbiota including psychrotrophic mi­ could be transferred from the epicarp to the pulp, or to other ready to eat
croorganisms (PM) or aerobic plate count (APC), coliforms (C) and yeast foods, representing a risk for consumers.
and molds (Y/M) on the surface of Hass avocados stored at 5 and 25 ◦ C
was simultaneously investigated. On avocados stored at 5 ◦ C, the initial Declaration of competing interest
PM, C and Y/M mean counts at day 0 were 6.6, 6.1 and 6.6 log CFU/
avocado, respectively, and remained with no significant changes (P > We wish to confirm that there are no known conflicts of interest
0.05) over time (Fig. 3). When avocados were stored at 25 ◦ C, the initial associated with this publication and there has been no significant
mean counts for APC, C and Y/M were 6.6, 6.1 and 6.6 log CFU/avo­ financial support for this work that could have influenced its outcome.
cado, respectively, and showed no significant changes over time (P > We confirm that the manuscript has been read and approved by all
0.05). The levels observed for these microbial groups are consistent with named authors and that there are no other persons who satisfied the
those previously reported for avocados collected at retail markets in criteria for authorship but are not listed. We further confirm that the
Mexico (García-Frutos et al., 2020) which presented mean counts of 6.2, order of authors listed in the manuscript has been approved by all of us.
5.4 and 5.1 log CFU/avocado for APC, C and Y/M, respectively. We confirm that we have given due consideration to the protection of
It is possible that these levels of background microbiota residing on intellectual property associated with this work and that there are no
the epicarp of fresh avocados contributed to create a hostile environ­ impediments to publication, including the timing of publication, with
ment for the inoculated pathogens, impeding their growth. It has been respect to intellectual property. In so doing we confirm that we have
demonstrated that the surfaces of fresh fruits and vegetables harbor followed the regulations of our institution concerning intellectual
large and diverse populations of bacteria that vary according to produce property.
types and cultivars, and to farming and storage conditions (Leff and
Fierer, 2013), which play an important role in maintaining the charac­ Acknowledgements
teristics of the products through competition, production of antimicro­
bial compounds or activation of plant defense mechanisms. In tomato This research was funded by the Mexican government program
plants that were inoculated with Salmonella Montevideo and S. Typhi­ “PRODEP - Convocatoria de Redes Temáticas de Colaboración” (project
murium, the presence of Enterobacter and Bacillus spp., negatively no. 103.5/16/12110), and the University of Guadalajara program
affected the persistence of the pathogen on the fruit (Shi et al., 2009), “Fortalecimiento de la Investigación y Posgrado 2019”. The authors
while in lettuce, an epiphytic isolate of Enterobacter asburiae decreased acknowledge the technical assistance of Nancy Mildred Rodríguez-
E. coli O157:H7 survival 20- to 30-fold on the foliage plant by compe­ Correa during the experiments of this project.
tition for carbon and nitrogen substrates (Cooley et al., 2006). Isolates of
Bacillus spp., Pseudomonas aeruginosa, P. fluorescens and yeasts recovered
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