FOODBORNE PATHOGENS AND DISEASE

Volume 17, Number 6, 2020

ª Mary Ann Liebert, Inc.
DOI: 10.1089/fpd.2019.2733

Occurrence of Pathogenic and Potentially Pathogenic

Bacteria in Microgreens, Sprouts, and Sprouted Seeds
on Retail Market in Riga, Latvia

Ieva Bergšpica,* Aija Ozola,* Elizabete Miltinxa, Laura Alksne, Ire

na Meistere,
Alla Cibrovska, and Lelde Grantinxa-Ievin xa
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Abstract
Microgreens and sprouts have been used for raw consumption for a long time and are generally viewed as a
healthy food. However, several serious outbreaks of foodborne illness have been recorded in European
countries, Japan, and North America. Many companies in Latvia nowadays are producing this type of products.
The aim of this study was to characterize the incidence of Shiga toxin–producing Escherichia coli (STEC),
Salmonella spp., and Listeria spp. in microgreens, sprouts, and seeds intended for domestic production of
microgreens on retail market in Riga, Latvia, from January to April 2019. The background microflora was
identified as well. A total of 45 samples were purchased, including fresh and processed sprouts, microgreens,
baby greens, as well as seeds intended for domestic production of microgreens and sprouts. The samples were
processed according to the methods set by the International Organization for Standardization (ISO)—ISO/TS
13136:2012 for STEC, ISO 6579-1:2017 for Salmonella spp., and ISO 11290-1:2017 for Listeria spp. Mole-
cular detection of Salmonella spp. was also performed using real-time polymerase chain reaction. The typical
and atypical colonies isolated from selective plates were identified with matrix-assisted laser desorption and
ionization time-of-flight mass spectrometry. Listeria monocytogenes was not detected in any of the tested
samples. However, the presence of Listeria innocua was detected in two (4.4%) of the samples. Three (6.7%)
samples of dried sprouts were positive for the STEC virulence genes. Salmonella spp. was detected in one
(2.2%) sample of common sunflower seeds. Altogether, 46 different background bacterial species were iden-
tified. The majority were environmental bacteria characteristic to soil, water, and plants, including coliform
bacteria. The results provide evidence that microgreens and seeds available for Latvian consumers are generally
safe, however, attention has to be paid to dried sprouts.

Keywords: microgreens, sprouts, seeds, STEC, Listeria spp., Salmonella spp.

Introduction seedlings in their youngest stage with fully developed cot-

yledons and emerging or partially expanded first pair of true

S prouts and microgreens are ready-to-eat specialty

crops that have grown in popularity as part of a healthy
diet due to their high nutritional value, as well as the fresh
flavors and visual appeal (Xiao et al., 2012; Paradiso et al.,
2018; Benincasa et al., 2019). Several terms are used for
these types of produce—‘‘sprouts,’’ ‘‘sprouted seeds,’’ and
‘‘microgreens’’—that often are used interchangeably. EU
has defined sprouts as a ‘‘product obtained from the germi-
nation of seeds and their development in water or another
medium, harvested before the development of true leaves,
and which is intended to be eaten whole, including the seed’’
(EC, 2013). Microgreens could be defined as ‘‘shoots or
leaves and they usually are cultivated in trays in growing
media or soil, indoors, or in greenhouses’’ (Xiao et al., 2014;
Kyriacou et al., 2016). Similar products to microgreens are
‘‘baby greens’’ that are harvested later than microgreens, but
before the plants have reached full maturity (Choe et al.,
2018).
These products are produced in warm and humid condi-
tions that are ideal for microbial growth. Microorganisms in
sprouts can originate from water, soil, air, animals, insects,
birds, and equipment. In addition, these products have a short
shelf life and are usually consumed without heat treatment or
decontamination procedures.

Institute of Food Safety, Animal Health, and Environment ‘‘BIOR,’’ Riga, Latvia.
*Both these authors contributed equally to this work.

1
 2 BERGŠPICA ET AL.
Salmonella spp. is the most common bacterial pathogen
responsible for outbreaks linked to fresh produce, whereas
leafy vegetables and sprouts are the most common food ve-
hicle implicated in these outbreaks (Callejón et al., 2015).
Different Escherichia coli strains have caused large out-
breaks with serious complications, and several of them have
been associated with sprouts (Watanabe et al., 1999; Buch-
holz et al., 2011; EFSA, 2011a; CDC, 2013). Another path-
ogen associated with fresh leafy produce is Listeria spp.
(Smith et al., 2018). Listeriosis outbreaks associated with
mung bean sprouts (CDC, 2015) as well as fruits (Buchanan
et al., 2017) have been reported recently. In response to the
increased number of outbreaks associated with sprouts, the
European Food Safety Authority (EFSA) has concluded that
these products pose microbial food safety concerns (EFSA,
2011b). Many studies have explored pathogen-associated
risks from the consumption of sprouts, but there is only a
limited number of studies about microgreens (Riggio et al.,
2019). The microbiological purity of microgreens can be
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affected by many factors, including the quality of growing
substrate (potting soil, compost, vermicompost, etc.) used for
plant growth (Muchjajib et al., 2015; Di Gioia et al., 2017;
Reed et al., 2018; Riggio et al., 2019). Vermicompost can
contain microorganisms that are human pathogens (Grantina-
Ievina et al., 2013). In addition, to the best of our knowledge,
no study of food safety aspects associated with the con-
sumption of sprouts and microgreens in Latvia or the nearest
Baltic or Nordic region has been documented so far.
The aim of this study was to characterize the incidence of
Shiga toxin–producing E. coli (STEC), Salmonella spp., and
Listeria spp., as well as the background microflora in mi-
crogreens, baby greens, sprouts, and seeds intended for the
production of microgreens available on the retail market in
Riga, Latvia, in one season of production from January 2019
to April 2019.
Materials and Methods
Sample collection
A total of 45 samples of microgreens, baby greens, and
seeds intended for domestic production of microgreens and
sprouts (fresh and processed) were purchased from different
stores, farmer’s market, as well as directly from the local
producers in Riga, Latvia, during the time period from Jan-
uary 2019 to April 2019. All samples were purchased and
processed before their expiration date and were stored until
processing under the appropriate conditions for the particular
Table 1. Sample Types Included and Tested in the Study
Type No/.
Microgreens 18
Seeds for the production of microgreens 9 9
Sprouts (fresh and processed) 12
Baby greens 6 6
Total 45
STEC, Shiga toxin–producing Escherichia coli.
product in separate single-use packaging, to avoid cross-
contamination. The following product-associated informa-
tion was recorded: type of product, species, date of sample
collection, manufacturer, the country of origin, and retailer.
The breakdown of all the sample categories included in the
study is shown in Table 1 and the overall characteristics of the
samples are listed in Table 2. The samples originated from 8
countries, 10 manufacturers, 6 local producers, and 5 retailers
and comprised 19 crop species. Most of the products were
locally produced. Several producers were using ecofriendly
technologies, vermicompost as fertilizer, recyclable sub-
strate, and reusable growing boxes.
Microbiological analysis
The incidence of STEC, Salmonella spp., and Listeria spp.
was assessed according to the methods set by the Interna-
tional Organization for Standardization (ISO)—ISO/TS
13136:2012 for STEC, ISO 6579-1:2017 for Salmonella spp.,
and ISO 11290-1:2017 for Listeria spp. (Fig. 1). All 45
samples were tested for the presence of STEC and Salmonella
spp., and 42 samples were tested for the presence of Listeria
spp. due to the insufficient amount of material in three
samples. A 25 g portion of each sample in five replicates for
each of the target bacteria were homogenized using a Sto-
macher 400 Circulator (Seward) for 30 s at 230 rpm in
225 mL of an appropriate enrichment medium. Modified
Trypticase Soy Broth (TSB) supplemented with 1 mL of
16 mg/L novobiocin solution per liter was used for STEC,
buffered peptone water was used for Salmonella spp., and
Half Fraser broth was used for Listeria spp. enrichment. The
samples were incubated for 24 h at 37C (for STEC and
Salmonella spp.) or 30C (for Listeria spp.).
After the incubation period, the STEC enrichment broth
was streaked on Tryptone Bile agar with X-glucoronide
(TBX) medium plates and incubated at 44C for 24 h. For
Salmonella spp. isolation, 0.1 mL of the enrichment suspen-
sion was added to 9 mL of Rappaport Vassiliadis medium
with soya (RVS), and 1.0–9 mL Muller-Kauffmann
Tetrathionate-novobiocin (MKTTn) broth, followed by in-
cubation of the samples for 24 h at 41.5C and 37C, re-
spectively. RVS and MKTTn broth samples were further
streaked on Xylose Lysine Deoxycholate agar (XLD) and
Brilliant Green Phenol Red Lactose Sucrose agar (BPLS)
plates and incubated at 37C for 24 h. For Listeria spp. iso-
lation, Half Fraser broth suspension was streaked on Agar
Listeria Ottavani & Agosti (ALOA) and Oxford agar plates,
as well as inoculated into 10 mL Fraser broth and incubated at
The number of samples tested for the presence
of particular bacteria (positive samples)
STEC Salmonella spp. Listeria spp.
18 (0) 18 (0) 16 (2)
(0) 9 (1) 8 (0)
12 (3) 12 (0) 12 (0)
(0) 6 (0) 6 (0)
45 (3) 45 (1) 42 (2)
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Table 2. The Overall Characteristics of Samples Included in the Study

Sample purchase Country
Sample date (dd.mm.year) Product type Species of origin Manufacturer Retailer
EM-1 24.01.2019 Seeds for the production Cress (Lepidum sativum L.) Lithuania Manufacturer 1 Supermarket chain 1
of microgreens
EM-2 24.01.2019 Microgreens Common sunflower (Helianthus annuus L.) Latvia Local producer 1a Supermarket chain 1
EM-3 24.01.2019 Microgreens Radish (Raphanus raphanistrum Latvia Local producer 1a Supermarket chain 1
L. subsp. sativus)
EM-4 30.01.2019 Microgreens Radish (R. raphanistrum L. subsp. sativus) Latvia Local producer 1a Supermarket chain 1
EM-5 30.01.2019 Microgreens Common sunflower (H. annuus L.) Latvia Local producer 1a Supermarket chain 1
EM-6 30.01.2019 Sprouts Garlic (Allium sativum L.) Sweden Manufacturer 2 Supermarket chain 1
EM-7 04.02.2019 Microgreens Arugula (Eruca sativa Mill.) Latvia Local producer 2a Local producer 2
EM-8 04.02.2019 Baby greens Pea (Pisum sativum L.) Latvia Local producer 2a Local producer 2
EM-9 10.02.2019 Microgreens Common sunflower (H. annuus L.) Latvia Local producer 1a Supermarket chain 1
EM-10 10.02.2019 Microgreens Arugula (E. sativa Mill.) Latvia Local producer 1a Supermarket chain 1
EM-11 10.02.2019 Microgreens Radish (R. raphanistrum L. subsp. sativus) Latvia Local producer 1a Supermarket chain 1
EM-12 10.02.2019 Sprouts (dried)b Rye (Secale cereale L.) Latvia Manufacturer 3 Supermarket chain 1
EM-13 10.02.2019 Sprouts (dried)b Common wheat (Triticum aestivum L.) Latvia Manufacturer 3 Supermarket chain 1
EM-14 10.02.2019 Sprouts (dried)b Oat (Avena sativa L.) Latvia Manufacturer 3 Supermarket chain 1
EM-15 13.02.2019 Baby greens Pea (P. sativum L.) Latvia Local producer 3 Local producer 3

3
EM-16 14.02.2019 Microgreens Common sunflower (H. annuus L.) Latvia Local producer 2a Local producer 2
EM-17 14.02.2019 Microgreens Radish (R. raphanistrum L. subsp. sativus) Latvia Local producer 2a Local producer 2
EM-18 14.02.2019 Sprouts Oat (A. sativa L.) Latvia Local producer 2a Local producer 2
EM-19 18.02.2019 Seeds for the production Common sunflower (H. annuus L.) Italy Manufacturer 4 Retail store 1
of microgreens
EM-20 18.02.2019 Seeds for the production Mung bean (Vigna radiata (L.) R. Wilczek), Switzerland Manufacturer 5 Retail store 1
of microgreens common lentil (Lens culinaris Medik.),
Radish (R. raphanistrum L. subsp. sativus)
EM-21 18.02.2019 Seeds for the production Cress (L. sativum L.) Czech Republic Manufacturer 6 Retail store 1
of microgreens
EM-22 24.02.2019 Sprouts (dried)b Oat (A. sativa L.) Latvia Manufacturer 3 Supermarket chain 1
EM-23 24.02.2019 Sprouts (dried)b Rye (S. cereale L.) Latvia Manufacturer 3 Supermarket chain 1
EM-24 24.02.2019 Sprouts (dried)b Common wheat (T. aestivum L.) Latvia Manufacturer 3 Supermarket chain 1
EM-25 05.03.2019 Microgreens Radish (R. raphanistrum L. subsp. sativus) Latvia Local producer 1a Supermarket chain 2
EM-26 05.03.2019 Baby greens Pea (P. sativum L.) Latvia Local producer 4 Supermarket chain 2
EM-27 24.03.2019 Seeds for the production Spelt wheat (Triticum spelta L.) Austria Manufacturer 7 Retail store 1
of microgreens
EM-28 24.03.2019 Seeds for the production Mung bean (V. radiata [L.] R. Wilczek) Czech Republic Manufacturer 8 Retail store 1
of microgreens

(continued)
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Table 2. (Continued)
Sample purchase Country
Sample date (dd.mm.year) Product type Species of origin Manufacturer Retailer
EM-29 24.03.2019 Seeds for the production Wheat (Triticum sp.) Austria Manufacturer 7 Retail store 1
of microgreens
EM-30 24.03.2019 Seeds for the production Mung bean (V. radiata [L.] R. Wilczek) Czech Republic Manufacturer 9 Retail store 1
of microgreens
EM-31 01.04.2019 Microgreens Common beet (Beta vulgaris L.) Latvia Local producer 1a Local producer 1
EM-32 01.04.2019 Microgreens Red cabbage (Brassica oleracea L.) Latvia Local producer 1a Local producer 1
EM-33 01.04.2019 Microgreens Brown mustard (Brassica juncea [L.] Czern.) Latvia Local producer 1a Local producer 1
EM-34 01.04.2019 Microgreens Broccoli (B. oleracea L. var. italica) Latvia Local producer 1a Local producer 1
EM-35 06.04.2019 Baby greens Arugula (E. sativa Mill.), Common beet Latvia Local producer 5 Farmer’s market 1
(B. vulgaris L.), Spinach
(Spinacia oleracea L.)
EM-36 06.04.2019 Baby greens Pea (P. sativum L.) Latvia Local producer 6 Farmer’s market 1
EM-37 07.04.2019 Microgreens Arugula (E. sativa Mill.) Latvia Local producer 4 Local producer 4

4
EM-38 07.04.2019 Microgreens Cress (L. sativum L.) Latvia Local producer 4 Local producer 4
EM-39 07.04.2019 Baby greens Pea (P. sativum L.) Latvia Local producer 4 Local producer 4
EM-40 07.04.2019 Microgreens Radish (R. raphanistrum L. subsp. sativus) Latvia Local producer 4 Local producer 4
EM-41 24.03.2019 Seeds for the production Oat (A. sativa L.) Czech Republic Manufacturer 8 Retail store 1
of microgreens
EM-42 09.04.2019 Sprouts (dried)b Common wheat (T. aestivum L.) Latvia Manufacturer 3 Supermarket chain 1
EM-43 09.04.2019 Sprouts Radish (R. raphanistrum L. subsp. sativus) Sweden Manufacturer 2 Supermarket chain 1
EM-44 16.04.2019 Sprouts (ground)c Wheat (Triticum sp.) China Manufacturer 10 Retail store 2
EM-45 16.04.2019 Sprouts (ground)c Barley (Hordeum vulgare L.) China Manufacturer 10 Retail store 2
a
Manufacturer used vermicompost as a component of soil.
b
Sprouts dried at temperatures up to 40C.
c
Sprouts were finely ground into powder.
 MICROFLORA OF SPROUTS AND MICROGREENS
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FIG. 1. Detection scheme for STEC, Salmonella spp. and
Listeria spp. STEC, Shiga toxin–producing Escherichia coli.
37C for 48 h. Single colonies from ALOA agar were
streaked onto Blood agar and incubated at 37C for 24 h to
detect the characteristic hemolytic activity of Listeria spp.
Colonies with typical STEC, Salmonella spp., and Listeria
spp. morphology from the respective selective plates as well
as at least five atypical colonies (if available) were streaked
on nutrient agar (NA) plates and incubated at 37C for 24 h
for further colony characterization.
Bacteria identification with matrix-assisted laser
desorption and ionization time-of-flight
mass spectrometry
Since atypical colonies were often observed on the E. coli,
Salmonella spp., and Listeria spp. selective plates, to identify
these bacteria, random colonies were transferred onto the NA
plates and analyzed with matrix-assisted laser desorption and
ionization time-of-flight (MALDI-TOF) mass spectrometry
(MALDI-TOF) using the MALDI Compass BioTyper 3
database (Bruker Daltonics).
Molecular detection of STEC and Salmonella spp.
Bacterial DNA was extracted from the enrichment broth
samples using InstaGene matrix (Bio-Rad) for STEC and
the mericon DNA Bacteria Kit (Qiagen) for Salmonella spp.
(Fig. 1). The presence of STEC was determined as described
in ISO 13136:2012 by real-time polymerase chain reaction
(PCR) with primers and probes for the STEC virulence genes
(stx1, stx2, eae), using ViiA 7 Real-Time PCR System
(Thermo Fisher Scientific) or Rotor-Gene Q real-time PCR
cycler (Qiagen) under the following conditions: 50C for
2 min, 95C for 10 min, followed by 40 cycles of 95C for
15 s and 60C for 1 min, ending with 60C for 30 s. Molecular
detection of Salmonella spp. was performed using the mer-
icon Salmonella spp. Kit (Qiagen) on a Rotor-Gene Q real-
time PCR cycler (Qiagen) under the following conditions:
95C for 5 min followed by 40 cycles of 95C for 15 s and
60C for 15 s, ending with 72C for 10 s.
5
Isolation of STEC
The samples for which the presence of stx1, stx2, or eae
genes was demonstrated were subjected to strain isolation to
confirm that the positive PCR signals were generated from
live E. coli bacteria. The enrichment broth was streaked on
CT-SMAC, MAC, and TBX plates that were incubated
overnight at 37C. Single colonies were streaked on NA
plates, pooled for DNA extraction, and incubated at 37C for
at least 6 h. The extraction of pooled colony DNA was per-
formed with PrepMan Ultra Sample Preparation Reagent
(Thermo Fisher Scientific). Pooled colonies were screened
for the STEC virulence genes using the same primers and
probes as described above. In the presence of positive signal
from the pool, single colonies from NA were screened to
determine the colony that possessed virulence genes.
Determination of E. coli serogroups
Serogroup detection was done according to the methods set
by EU Reference Laboratory for E. coli (EU-RL-VTEC).
Serogroups O26, O103, O111, O145, and O157 were tested
with real-time PCR on Rotor-Gene Q real-time PCR cycler
according to the EU-RL-VTEC Method 11 (EU-RL-VTEC,
2018), serogroup O104 was examined with PCR on ProFlex
PCR System (Applied Biosystems) according to the EU-RL-
VTEC Method 03 (EU-RL-VTEC, 2014), and PCR products
were visualized on QIAxcel multicapillary electrophoresis
instrument (Qiagen).
Illumina sequencing and data analyses
To gain a more detailed insight into the genomic profile of
stx1-producing STEC isolate, a full genome sequence of that
isolate was determined. DNA library was constructed with the
Nextera XT DNA Library Preparation Kit (Illumina). The li-
brary was sequenced on a MiSeq System (using MiSeq Re-
agent Kit v3 (Illumina) and 300 bp long paired reads were
obtained. Quality measures of sequences were analyzed with
the FastQC program (version 0.11.8) (Andrews, 2010). For
genome de novo assembly, Velvet 1.1.04 (Zerbino and Birney,
2008) was used and the whole genome sequence was obtained
with 79 · coverage. The characterization of stx1-producing
STEC strain was performed with the online tools Virulen-
ceFinder 2.0 ( Joensen et al., 2014) and SerotypeFinder 2.0
( Joensen et al., 2015) developed by the Center for Genomic
Epidemiology of the Technical University of Denmark. The
raw reads generated were submitted to the European Nucleo-
tide Archive with the study accession number PRJEB33523.
Results
The presence of target pathogenic bacteria assessed
by microbiological methods
The target pathogenic bacteria Salmonella spp. and Lis-
teria monocytogenes were not detected in any of the tested
samples according to the standard methods ISO 6579-1:2017
and ISO 11290-1:2017, respectively.
The presence of target pathogenic bacteria assessed
by molecular analyses
All 45 samples were tested for the presence of STEC (ISO
13136:2012) and Salmonella spp.-specific genes using real-
 6 BERGŠPICA ET AL.
time PCR. Three (6.7%) samples (dried sprouts of common
wheat EM-13 and EM-24, and rye EM-23) were positive for
the E. coli virulence genes. Shiga toxin-coding gene stx1 was
present in two samples (EM-23, EM-24), stx2 in one sample
(EM-24), and intimin-coding gene eae in two samples (EM-
13, EM-24), with the sample EM-24 found positive for all
three E. coli virulence genes. MALDI-TOF analyses con-
firmed the presence of E. coli in all three of these samples.
Thus, the presence of STEC virulence genes was detected in
three out of seven (42.9%) E. coli-positive samples and all
three positive samples originated from dried sprouts.
Samples that were positive for E. coli virulence genes
(EM-13, EM-23, EM-24) were subjected to molecular de-
tection of serogroups and strain isolation. We isolated an
stx1-positive strain from sample EM-24 and determined its
whole genome sequence. Genome analysis showed that the
sample EM-24 contained the E. coli serotype O11:H48. In
addition, by using VirulenceFinder 2.0 we determined that
this strain harbored the stx1d subtype. EM-13 was found to
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contain the serogroups O26 and O157, but EM-23 contained
the serogroup O145. However, strain isolation for these
samples was not successful, therefore we were not able to
confirm serogroups in colonies.
None of the samples was found to contain Salmonella spp.-
specific genes according to real-time PCR.
Identification of background bacteria
Since atypical colonies were often observed on the E. coli,
Salmonella spp., and Listeria spp. selective plates, random
colonies were transferred to the NA plates and analyzed with
MALDI-TOF mass spectrometry. Altogether, 46 different
bacteria species from 22 genera and 12 families were identified
(Table 3). E. coli was detected in seven (15.5%) of the samples
and Salmonella spp. in one (2.2%) sample (EM-19). We did
not find L. monocytogenes in any of the samples, although the
presence of another Listeria genus species, Listeria innocua,
was detected in two (4.4%) samples (EM-4 and EM-9).
Among the rest of the bacteria detected with MALDI-TOF, the
majority were environmental bacteria characteristic to soil,
water, and plants, including coliform bacteria (Table 3).
Discussion
Nowadays there is a growing interest in healthy lifestyle
and the nutritional value and quality of food. Microgreens
and sprouts are attractive novelty products that offer both
high nutritional value and esthetic qualities (Choe et al.,
2018; Paradiso et al., 2018; Benincasa et al., 2019). However,
sprout and microgreen manufacturing conditions facilitate
the growth of microorganisms that can pose serious food
safety risks. Overall, fresh vegetables and juices were asso-
ciated with 3.8% of foodborne outbreaks (5.1% of the total
number of cases) in the EU in 2017 (EFSA and ECDC, 2018)
and, over the years, sprouts have been implicated in several
serious outbreaks in different countries (Watanabe et al.,
1999; Buchholz et al., 2011; Callejón et al., 2015). Therefore,
it is vital to monitor and evaluate the microbial risks arising
from these products. This study characterizes the presence of
pathogenic and potentially pathogenic bacteria in various
types of sprouts, microgreens, and baby greens in Latvia. One
of the limitations of the study was that the samples were
purchased only in Riga, Latvia. However, the retail market of
Riga is supplied by the local producers located in various
regions of the country and thus the retail market of Riga
represents the overall situation in Latvia.
In general, the target pathogenic bacteria were detected in
a minority of the samples. The presence of E. coli stx genes
was detected in three dried sprout samples that were prepared
by drying sprouted grains at 40C. All of these samples were
produced by the same local manufacturer and distributed by
one supermarket chain. We isolated the stx1-containing
E. coli strain from one of the samples (EM-24). While the
DNA isolated from the enrichment broth of this sample also
showed the presence of stx2 and eae genes, we did not detect
the presence of stx2 and eae genes in the isolated colony.
Thus, we confirmed the presence of pathogenic E. coli in one
(2.2%) of the analyzed samples, as well as revealed two other
highly suspicious samples that harbored the STEC pathoge-
nicity genes. Although we were not able to prove the presence
of live STEC cells in these two samples and the origin of the
STEC genes might be from nonviable cells that do not pose
harm, the presence of STEC pathogenicity genes in these
products is alarming, since according to ISO/TS 13136:2012
such results must be interpreted as a presumptive detection of
STEC. The range of STEC-positive samples among EU
countries reporting the results from sprouted seeds on retail
market is between 0% and 0.3% for the time period of 2013–
2017 (EFSA and ECDC, 2018), which is lower than in the
present investigation.
DNA isolated from the enrichment broth of sample EM-13
showed the presence of different serogroups (O26, O157),
whereas sample EM-23 showed O145 serogroup-specific
genes. However, the isolation of STEC was unsuccessful
in both cases, which could be explained by the low number of
viable bacteria in the sample. To our knowledge, no cases
of illness have been attributed to E. coli contamination of
sprouts or microgreens in Latvia during the study period, but
there has been one registered case of STEC infection in the
time period from January to April 2019 (CDPC, 2019), al-
though, to the best of our knowledge, this infection has not
been linked to the consumption of sprouts.
By using MALDI-TOF we identified the presence of
E. coli in 7 (15.7%) out of a total of 45 samples, including 5
dried sprout samples.
In two samples of microgreens, namely, radish (EM-4) and
common sunflower (EM-9), the presence of L. innocua was
detected with MALDI-TOF mass spectrometry method and
in one sample of common sunflower microgreens (EM-19)
the presence of Salmonella spp. was detected with MALDI-
TOF mass spectrometry. The most common foodborne
pathogenic bacteria of Listeria genus is L. monocytogenes,
although to a much lower extent than Salmonella spp. and
E. coli. However, a large U.S. foodborne pathogen study
found that the prevalence of L. monocytogenes in sprouts, as
well as other vegetables and fruits, was even higher than that
of Salmonella spp. and E. coli (both O157:H7, and non-
O157), probably due to the fact that L. monocytogenes can
grow under refrigerated conditions (Zhang et al., 2018). In
general, L. innocua is considered to be a nonpathogenic
Listeria species; however, a recent study demonstrated that
the atypical hemolytic L. innocua is virulent and closely re-
lated to L. monocytogenes (Moura et al., 2019).
Among the rest of the bacteria detected with MALDI-TOF,
the majority were environmental bacteria characteristic to
 Table 3. Bacteria Identified in Microgreens, Baby Greens, Seeds, and Sprouts with Matrix-Assisted Laser
Desorption and Ionization Time-of-Flight Mass Spectrometry
Family Genus Species Samples
Bacillaceae Bacillus Bacillus cereus EM-5, EM-9, EM-15, EM-41
Bacillus circulans EM-1, EM-5, EM-15, EM-17, EM-41,
EM-44, EM-45
Bacillus licheniformis EM-25, EM-30, EM-42, EM-44, EM-45
Bacillus oleronius EM-44
Bacillus pumilus EM-1, EM-20, EM-29, EM-30,
EM-41, EM-44
Lysinibacillus Lysinibacillus boronitolerans EM-29
Lysinibacillus fusiformis EM-17
Burkholderiaceae Cupriavidus Cupriavidus gilardii EM-40
Corynebacteriaceae Corynebacterium Corynebacterium callunae EM-38, EM-39, EM-40
Enterobacteriaceae Citrobacter Citrobacter amalonaticus EM-29
Citrobacter braakii EM-2, EM-8, EM-16, EM-18, EM-24,
EM-32, EM-35, EM-36, EM-37,
EM-38, EM-39, EM-40, EM-42
Citrobacter freundii EM-3, EM-6, EM-7, EM-8, EM-17,
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EM-18, EM-24, EM-25, EM-37,

EM-38, EM-41, EM-43
Citrobacter youngae EM-43
Cronobacter Cronobacter sakazakii EM-12, EM-16, EM-22, EM-23, EM-34
Enterobacter Enterobacter asburiae EM-19, EM-27, EM-29, EM-31,
EM-33, EM-35
Enterobacter cloacae EM-5, EM-19, EM-21, EM-28, EM-31,
EM-32, EM-42
Enterobacter kobei EM-31, EM-32, EM-33
Enterobacter ludwigii EM-25
Escherichia Escherichia coli EM-4, EM-8, EM-12, EM-14, EM-23,
EM-24, EM-42
Escherichia hermannii EM-26
Hafnia Hafnia alvei EM-18, EM-37
Klebsiella Klebsiella oxytoca EM-8, EM-43
Klebsiella pneumoniae EM-4, EM-8, EM-11, EM-14, EM-16,
EM-24, EM-26, EM-33, EM-38,
EM-42, EM-43
Klebsiella variicola EM-22, EM-26, EM-34, EM-43
Leclercia Leclercia adecarboxylata EM-8
Raoultella Raoultella ornithinolytica EM-8
Raoultella planticola EM-16, EM-35
Raoultella terrigena EM-8, EM-39
Salmonella Salmonella sp. EM-19
Enterococcaceae Enterococcus Enterococcus casseliflavus EM-9, EM-42
Enterococcus faecium EM-9, EM-12, EM-13, EM-16, EM-42
Enterococcus hirae EM-12
Enterococcus mundtii EM-16, EM-18, EM-22, EM-23,
EM-30, EM-42
Listeriaceae Listeria Listeria innocua EM-4, EM-9
Microbacteriaceae Curtobacterium Curtobacterium sp. EM-36
Microbacterium Microbacterium arborescens EM-15, EM-30, EM-37, EM-38,
EM-39, EM-40, EM-42
Microbacterium testaceum EM-36
Micrococcaceae Arthrobacter Arthrobacter woluwensis EM-6, EM-42, EM-43
Moraxellaceae Acinetobacter Acinetobacter baumannii EM-16
Promicromonosporaceae Cellulosimicrobium Cellulosimicrobium cellulans EM-34
Pseudomonadaceae Pseudomonas Pseudomonas aeruginosa EM-8, EM-16, EM-17
Pseudomonas balearica EM-19
Pseudomonas flavescens EM-41
Pseudomonas guariconensis EM-34
Pseudomonas putida EM-30, EM-41
Staphylococcaceae Staphylococcus Staphylococcus hominis EM-1
Entries in bold represent genera of coliform bacteria.

7
 8 BERGŠPICA ET AL.
soil, water, and plants, including coliform bacteria (Table 3).
Some of them were well-known foodborne pathogens such as
Bacillus cereus (Bottone, 2010), as well as bacteria that are
infectious to humans, but not typically associated with
foodborne infections, including Citrobacter braakii (Oyeka
and Antony, 2017), Citrobacter freundii (Whalen et al.,
2007), Citrobacter youngae (Chen et al., 2013), Escherichia
hermannii (Ioannou, 2019), Cronobacter sakazakii (Singh
et al., 2015), Pseudomonas aeruginosa (Azam and Khan,
2019), Pseudomonas putida (Kim et al., 2012), and Kleb-
siella pneumoniae (Navon-Venezia et al., 2017).
Conclusions
The study results provide evidence that microgreens and
seeds marketed in Latvia for the domestic production of
microgreens are generally safe, however, there is some
concern with regard to dried sprouts, as E. coli virulence
genes were identified in three samples. Further studies are
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required to monitor the situation.
Author Contributions
I.B. designed and conducted the study, interpreted data,
prepared the article; A.O. summarized data, interpreted data,
and prepared the article; E.M. performed microbiological and
molecular biology analyses; L.A. performed MALDI-TOF
analyses; I.M. performed Illumina sequencing and data an-
alyses; A.C. coordinated microbiological studies; L.G.-I.
designed and supervised the study, interpreted data, and
prepared the article.
Disclosure Statement
No competing financial interests exist.
Funding Information
This research was cofinanced by European Regional De-
velopment Fund (ERDF) (85%) and the state budget of Latvia
(7.5%) under the project number 1.1.1.1/16/A/258 ‘‘Devel-
opment and the application of innovative instrumental ana-
lytical methods for the combined determination of a wide
range of chemical and biological contaminants in support of
the bio-economy in the priority sectors of economy.’’
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Address correspondence to:
Aija Ozola, PhD
Institute of Food Safety, Animal Health
and Environment ‘‘BIOR’’
3 Lejupes Street
Riga LV-1076
Latvia
E-mail: aija.ozola@bior.lv