Food Protection Trends - July/August 2021 - 417

sakazakii), is considered an emerging foodborne pathogen,
and plant material is considered its highly probable vehicle
of transmission (14). Because this organism is ubiquitous in
the environment (7), it may survive for long periods in dry
foods, making it a pathogen of concern (22). Cronobacter is
listed among pathogens under the category " severe hazard
for restricted populations, life threatening or substantial
chronic sequelae or long duration " (24). Because LMFs
such as cereals, grains, and spices are widely traded food
commodities (13), surveillance of the global LMF supply
chain for Cronobacter contamination is important. Mauritius
is a net food importer (25), importing many essential food
requirements, including LMF commodities. However, to
our knowledge no studies have been conducted on the
occurrence of Cronobacter spp. in LMFs in Mauritius. The
objectives of this study were (i) to determine the prevalence
of Cronobacter contamination in the global LMF supply chain
through use of an online surveillance database and (ii) to
assess the prevalence of Cronobacter in LMFs locally available
in Mauritius.
MATERIALS AND METHODS
Notifications of Cronobacter contamination worldwide
HorizonScan (version 2.1.6, FERA Science, Sand Hutton,
York, UK) was the online database of choice for this study of
global food contamination issues. This food safety database is
a global surveillance and tool for tracking contamination and
adulteration of food ingredients and products in international
commerce. The database was used to retrieve a history of
recalls or other notifications of Cronobacter contamination
of food products. To perform an unrestricted search and to
obtain the maximum number of notifications, " Cronobacter "
was the search term, the option " all data " was selected, and all
other fields were purposely left blank.
Sampling and aw determination of commercially
available LMFs
Samples of LMFs (n = 302) for 10 broad food categories
(cereals, dried adult milk, dried noodles, flour, infant foods,
nuts, pulses, spices, seasonings, and tea) were purchased from
supermarkets in Mauritius. The samples were kept in their
original packaging at room temperature away from sunlight
to prevent degradation or change in the microbiota of the
food. The aw
of the samples was determined with a calibrated
aw meter (Novasina, Lachen, Switzerland) with triplicate
measurements and two independent replicates.
Microbiological analysis of LMFs for Cronobacter spp.
Cronobacter spp. isolates were recovered according to the
protocol of Berthold-Pluta et al. (6), and the sensitivity of
the method was improved by increasing the analytical sample
size from 10 to 25 g. The 25-g samples of each product were
weighed aseptically, mixed with 225 mL of buffered peptone
water, and incubated at 37°C for 18 to 24 h. The primary
mixture was then enriched in E. sakazakii enrichment broth
(26) for 24 h, plated on selective and chromogenic HiCrome
E. sakazakii agar (HiMedia, Mumbai, India), and incubated
at 37°C for 24 h. The selectivity of the chromogenic medium
is based on the activity of α-glucosidase, which is produced
by all strains of Cronobacter (16). Deep blue colonies
were streaked onto tryptic soy agar (TSA; HiMedia) and
incubated at 26°C for up to 72 h. Yellow colonies on TSA
were suspected to be Cronobacter spp. and were picked
up for Gram staining and microscopic examination. Pale
yellow colonies that appeared after 72 h were incubated for
another 24 h until colonies appeared deep yellow. According
to Garbowska et al. (16), the yellow pigmentation is
characteristic of the majority of Cronobacter strains. Growth
of yellow colonies on TSA is currently used as a criterion for
identification of presumptive E. sakazakii isolates (29), but
the intensity of pigmentation differs among strains (12).
Molecular identification of presumptive Cronobacter isolates
Presumptive Cronobacter isolates were identified
based on the formation of uniform deep blue colonies on
HiCrome E. sakazakii agar and intense yellow colonies
on TSA. Five isolates from wheat flour, black pepper,
masala powder, black tea powder, and dried milk were
randomly selected for further identification by a PCR
assay. Genomic DNA was extracted according to the
phenol-chloroform protocol of Ausubel et al. (5) followed
by DNA quantification by UV spectrophotometry
(Spectronic 1201, Milton Roy, Lenexa, KS). For PCR
identification of the isolates at the genus level, a pair
of primers were designed targeting a 438-bp fragment
of the gyrase B gene (gyrB) of Cronobacter: gyrB-F:
5′-ATGGATAAAGAGGGCTACAG-3′ and gyrB-R:
5′-GCCTGATTCTTACGGTTAC-3′ (1, 2). The PCR
thermocycling program was predenaturation at 95°C for
5 min; amplification in 35 cycles of 94°C for 30 s, 62°C
for 30 s, and 72°C for 30 s; and final elongation at 72°C
for 10 min (8). The amplification products were analyzed
by electrophoresis on a 1.5% agarose gel with a DNA
size ladder. The gel was stained with ethidium bromide,
visualized with a UV transilluminator, and photographed.
PCR products for the gyrB gene were subsequently sent
to Inqaba Biotec (Pretoria, South Africa) for purification
and sequencing with the Sanger dideoxy sequencing
technology. The forward and reverse sequences obtained
from Inqaba Biotec were processed with the BioEdit
sequence alignment editor (version 7.0.5.3, Informer
Technologies, https://www.informer.com/) to yield
a consensus sequence. The consensus sequences were
verified and manually corrected where needed. A BLAST
search on the NCBI website (https://blast.ncbi.nim.
nih.gov/Blast.cgi) was done to compare the partial gyrB
sequence with sequences in the NCBI GenBank database.
July/August Food Protection Trends 417
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