International Journal of Food Microbiology 263 (2017) 38–46

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International Journal of Food Microbiology

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

Occurrence of transferable antibiotic resistances in commercialized ready- MARK

to-eat mealworms (Tenebrio molitor L.)
Andrea Osimani, Federica Cardinali, Lucia Aquilanti⁎, Cristiana Garofalo, Andrea Roncolini,
Vesna Milanović, Marina Pasquini, Stefano Tavoletti, Francesca Clementi
Dipartimento di Scienze Agrarie, Alimentari ed Ambientali, Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, Italy

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

Keywords: The present study aimed to assess the occurrence of transferable determinants conferring resistance to tetra-
Novel foods cyclines, macrolide-lincosamide-streptogramin B, vancomycin, beta-lactams, and aminoglycosides in 40 samples
Risk assessment of commercialized edible mealworms (Tenebrio molitor L.) purchased from European Union (EU) and non-EU
Edible insects producers. A high prevalence of tet(K) was observed in all of the samples assayed, with percentages of PCR-based
Tetracycline
positivity that ranged from 80% (samples from Thailand) to 100% (samples from the Netherlands, Belgium and
Antibiotic resistance determinants
France). For macrolides, erm(B) prevailed, being detected in 57.5% of the samples assayed, whereas erm(A) and
erm(C) were detected with lower frequencies. Genes for resistance to vancomycin were only detected in samples
produced in France and Belgium, with 90% and 10% of the samples being positive for vanA, respectively.
Beta-lactamase genes were found with low occurrence, whereas the gene aac-aph, conferring high resistance
to aminoglycosides, was found in 40% of the samples produced in the Netherlands and Belgium and 20% of the
samples produced in Thailand. The results of Principal Coordinate Analysis and Principal Component Analysis
depicted a clean separation of the samples collected from the four producers based on the distribution of the 12
AR determinants considered. Given the growing interest on the use of mealworms as a novel protein source, AR
detection frequencies found in the present study suggest further investigation into the use of antibiotics during
rearing of this insect species and more extensive studies focused on the factors that can affect the diffusion of
transferable ARs in the production chain. Until such studies are completed, prudent use of antibiotics during
rearing of edible insects is recommended.

1. Introduction can be considered a good, environmentally friendly alternative to

conventional food production animals.
The World Health Organization (WHO) reports that 800 million Worldwide, 2.5 billion people include edible insects in their diet
people are chronically undernourished. In particular, it is estimated (van Huis et al., 2013). The most consumed species belong to the orders
that over 40 million preschool-age children were moderately acutely Coleoptera, Hymenoptera, Isoptera, Lepidoptera, Orthoptera, and Dip-
malnourished (WHO, 2012). In addition to this concerning data, the tera; among these, Vespula spp., Macrotermes spp., Encosternum spp.,
demand for meat and dairy products in developing countries is growing Oxya spp., Rhynchophorus phoenicis, Oecophylla smaragdina, Acheta do-
dramatically. Such increasing demand for high-quality proteins requires mesticus, Gonimbrasia bellina, Apis mellifera, Bombyx mori, Tenebrio mo-
intense farming of meat and dairy animals that has become difficult to litor, and Hermetia illucens are currently reared for human and animal
sustain due to the high environmental impact (Klunder et al., 2012; consumption or simply harvested from the wild (Payne et al., 2016).
Makkar et al., 2014; van der Spiegel et al., 2013; van Huis et al., 2013; Edible insects can be consumed dried (with or without lyophilisation),
Yen, 2009). fried, boiled, or processed into powder to be used as ingredient for
Edible insects or foods containing processed edible insects can be further preparations (i.e., bakery products, food supplements)
considered a notable food resource, since they are rich in high-quality (Grabowski and Klein, 2017).
proteins and present valuable characteristics in both the lipid fractions Although in Europe the use of edible insects as food is still far from
and amino acid profiles (Osimani et al., 2017a; Rumpold and Schlüter, commonplace, a recent study by Verbeke (2016) revealed that, in
2013). Moreover, as reported by Oonincx et al. (2015), edible insects Western societies, younger males with little interest in meat could be

⁎
Corresponding author.
E-mail address: l.aquilanti@univpm.it (L. Aquilanti).

http://dx.doi.org/10.1016/j.ijfoodmicro.2017.10.009
Received 30 March 2017; Received in revised form 6 September 2017; Accepted 3 October 2017
Available online 05 October 2017
0168-1605/ © 2017 Elsevier B.V. All rights reserved.
A. Osimani et al. International Journal of Food Microbiology 263 (2017) 38–46

the early adopters of insects as a novel food. However, in the European aph] was assessed through optimized PCR and nested PCR assays. These
Union, there is still a lack of regulatory guidelines regarding the pro- determinants correspond to those most frequently detected in both
duction, processing and distribution of edible insects. Beginning Jan- foodborne human pathogens (Rolain, 2013) and commensal bacteria
uary 1, 2018, Regulation (EU) No 2015/2283 of the European Parlia- (Aarts and Margolles, 2015; Devirgiliis et al., 2011).
ment and of the Council on novel foods will take effect. This newly
issued regulation includes insects and insect components in the novel
foods category. Moreover, the same regulation introduced a simplified 2. Materials and methods
procedure for placing traditional foods from non-EU countries on the
EU market. 2.1. Purchase of edible insects
The growing interest in edible insects has triggered new and more
extensive research on aspects related to their microbiological safety, Forty samples of edible mealworms (boiled, dried and salted) were
though these studies frequently have a relatively limited number of provided by four dealers located in the Netherlands (samples NL1 to
samples (Ali et al., 2010; Klunder et al., 2012; Osimani et al., 2017a; NL10), Thailand (samples TH1 to TH10), Belgium (samples BL1 to
Stoops et al., 2016; Vandeweyer et al., 2017). BL10) and France (samples FR1 to FR10). The samples were provided in
From the available scientific literature, the main risk associated sealed plastic boxes of varying weights and delivered via international
with the consumption of insects comes from the microorganisms re- transport. They were analyzed within their shelf life at ambient tem-
siding in their intestinal tract or those derived from the insect en- perature, which ranged from nine to twelve months, depending on the
vironment, and hence dependent on the rearing conditions, processing producer.
and storage (ANSES Opinion, 2015). Additionally, microorganisms, and
especially pathogens, can carry resistance to antibiotics commonly used
2.2. Microbial enumeration
in clinical and veterinary practices.
Antibiotic resistance (AR) represents one of the major threats to
Ten grams of each sample was crushed with a mortar under sterile
public health and food safety worldwide. In 2011, the Codex
conditions followed by 10 min of homogenization at 260 rpm in 90 mL
Alimentarius Commission (CAC, 2011) issued guidelines for risk ana-
of peptone-saline solution with a Stomacher 400 Circulator apparatus
lysis of foodborne antimicrobial resistance aimed to assess the risk to
(VWR International, Milan, Italy). The resulting suspensions were di-
human health related to the occurrence of transmissible antibiotic re-
luted 10-fold and subjected to microbial enumeration of total meso-
sistant microorganisms and AR determinants in food and animal feed.
philic aerobes, Enterobacteriaceae, lactic acid bacteria, and spore
The World Health Organization (WHO), the Food and Agriculture Or-
forming bacteria in appropriate growth media by inclusion spreading as
ganization of the United Nations (FAO), and the World Organization for
already described by Osimani et al. (2017a). All samples were analyzed
Animal Health (OIE) are working to prevent the spread of antibacterial
in duplicate.
resistance with a global action plan aimed at optimizing the use of
antibiotics in human clinical practice and animal farming (WHO,
2016). 2.3. Bacterial DNA extraction
In insect rearing, antibiotics are used only as emergency treatment
(Eilenberg et al., 2015; Tarapoulouzi et al., 2013). As early as 1992, Microbial DNA, directly extracted from the mealworms, was iso-
Peng et al. (1992) described the use of chlortetracycline in laboratory- lated as described by Osimani et al. (2017b). Briefly, the PowerFood
reared honey bees, and two decades later, Hirose et al. (2006) proposed Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, USA)
the use of streptomycin to prolong the life of reared Nezara viridula. was used, followed by quantity and purity assessment via optical
More recently, Cappellozza et al. (2011) reported the use of chlor- readings at 260, 280 and 234 nm using a UV–Vis Shimadzu UV-1800
amphenicol in the rearing of silkworms to reduce the loss of cocoons. spectrophotometer (Shimadzu, Kyoto, Japan) (Garofalo et al., 2015).
The presence of antibiotics (netilmicin and streptovaricine G) as con-
taminants has also recently been highlighted in a number of edible
insects commercialized in Belgium for human consumption, including 2.4. PCR and nested PCR amplification of AR determinants
Galleria mellonella, Locusta migratoria, Acheta domesticus and Alphitobius
diaperinus (Poma et al., 2017), thus representing a risk for human The DNA extracts were subjected to a first set of PCR reactions,
health. targeting the 12 selected AR determinants; negative samples were fur-
As reported by Larson et al. (2008), pest insects can be vectors of ther subjected to a set of nested PCR reactions to improve amplification
antibiotic resistant microorganisms, and more recently, it has been re- sensitivity.
ported that the intestinal tract of cockroaches can represent an in vivo PCR and nested PCR reactions were carried out as previously de-
model for the natural conjugation-mediated horizontal transfer of AR scribed by Osimani et al. (2017b). Briefly, 2 μL of DNA extract (~ 50 ng
plasmids among bacteria (Anacarso et al., 2016). The exchange of genes of bacterial DNA) or PCR product was amplified in a reaction mixture
in the insect gastrointestinal tract has also been reported by Crippen that was composed of 1× buffer, 50 pmol each primer, 0.2 mM dNTPs
and Poole (2009), where horizontal transfer of AR genes in Alphitobius (2.5 mM for the amplification of erm genes in both assays) and 0.75 U
diaperinus (Coleoptera: Tenebrionidae) larvae was described. Taq polymerase. Thermal cycling conditions and primers used for PCR
To date, only scarce data regarding the occurrence and distribution and nested PCR assays are reported in Tables S1 and S2 (Supplementary
of AR genes in edible insects are available, and the only two published material). In Table 1, reference cultures used as positive and negative
studies have highlighted that this novel food can effectively constitute a controls are listed. PCR mixture supplemented with water instead of
reservoir of transferable resistances, thereby suggesting the need for DNA was used as a blank. A PCR workstation with a built-in UV source
further investigation (Milanović et al., 2016; Osimani et al., 2017b). (DNA/RNA UV-Cleaner UVC/T-M-AR, Biosan, Riga, Latvia) was used
Based on these premises, this study was aimed to assess the occur- for the preparation of reaction mixtures.
rence of transferable AR determinants in 40 samples of edible meal- All the amplifications were carried out in a MyCycler thermal cycler
worms (Tenebrio molitor L.) marketed in the EU. The prevalence of 12 (Bio-Rad Laboratories, Hercules, USA). PCR products were analyzed
selected determinants conferring resistance to tetracyclines [tet(M), tet under UV light using the Complete Photo XT101 system (Explera, Jesi,
(O), tet(S), tet(K)], macrolide-lincosamide-streptogramin B (MLSB) [erm Italy) as reported by Milanović et al. (2016). False-positive results and
(A), erm(B), erm(C)], vancomycin (vanA, vanB), beta-lactams (blaZ, contamination in PCR assays were minimized by adopting basic pre-
mecA) and aminoglycosides [aac(6′)-Ie aph(2″)-Ia, abbreviated as aac- cautions suggested by Borst et al. (2004).

39
A. Osimani et al. International Journal of Food Microbiology 263 (2017) 38–46

Table 1 Table 2
Bacterial reference strains used in this study. ANOVA results for total mesophilic aerobes and spore-forming bacteria (log cfu g− 1) in
40 samples of edible mealworms according to producer.
Bacterial reference strain AR gene
Source of variation df Total mesophilic aerobes Spore-forming bacteria
Staphylococcus aureus M.P. erm(A)a
Enterococcus hirae Api 2.16 erm(B)a Mean squares F Mean squares F
Staphylococcus spp. SE12 erm(C)b
⁎⁎⁎
Enterococcus faecium PF3U vanAb Producer 3 59.528 68.61 34.401 38.05⁎⁎⁎
Enterococcus faecalis ATCC 51299 vanBc Samples [producer] 36 0.868 13.05⁎⁎⁎ 0.904 3.90⁎⁎⁎
Lactobacillus casei/paracasei ILC2279 tet(M)b Error 40 0.066 0.232
Streptococcus pyogenes 7008 tet(O)a
Enterococcus italicus 1102 tet(S)b
Staphylococcus aureus COL. tet(K)a
Producer Total mesophilic aerobes Spore-forming bacteria
Staphylococcus aureus 27R mecAb
Staphylococcus aureus ATCC 2921 blaZc
Mean ± SD Mean ± SD
Enterococcus faecium M48 aac-apha
Enterococcus faecalis JH2-2 Negative controld c
The Netherlands 3.14 ± 0.30 2.57 ± 0.34b
a
Thailand 1.32 ± 0.96d 0.79 ± 1.06c
Culture collection of Dipartimento di Scienze della Vita e dell'Ambiente (DiSVA),
Belgium 4.05 ± 0.19b 3.95 ± 0.27a
Università Politecnica delle Marche, Italy. France 5.43 ± 0.86a 1.99 ± 0.94b
b
Culture collection of Dipartimento di Scienze Agrarie, Alimentari ed Ambientali
(D3A), Università Politecnica delle Marche, Italy. cfu colony forming units.
c
ATCC, American Type Culture Collection, Manassas, U.S.A. df degrees of freedom.
d
Jacob and Hobbs (1974). SD standard deviation.
Within each column, means followed by different letters are significantly different
2.5. Statistical analysis (P < 0.05).
⁎⁎⁎
Significant P < 0.0001.

The results of the viable counts were expressed as the mean of the
log colony forming units (cfu) per gram of sample ± standard devia- the number of PCR- or nested PCR-positive samples divided by whole
tion. Given the high number of samples showing microbial counts set of 40 samples.
below 1 log cfu g− 1 for some microbiological parameters, two different
statistical approaches were adopted. Briefly, viable counts of total 3. Results and discussion
mesophilic aerobes and spore forming bacteria (≥1 log cfu g− 1
in > 50% of the samples) were subjected to one way analysis of var- In this study, 40 samples of edible mealworm larvae purchased from
iance (ANOVA) with the following sources of variation: “Producer” (P), both EU and non-EU countries have been subjected to viable counting
“Samples within Producer” (S[P]) and “error” (JMP 11.0.0, SAS Institute of various microbial groups and further screened for the occurrence of
Inc., Cary, NC, USA). The variance among Producers was tested using 12 transferable resistance genes. Among edible insects, mealworm
“the among Samples within Producers variance” as denominator of the larvae are easy to breed and represent a very promising source of
F test. The “among Samples within Producers” variance was tested protein and fats, providing 20% and 15% of these nutrients, respec-
using the error mean squares as denominator of the F test. Multiple tively (Gasco et al., 2016; Zhao et al., 2016). In some EU countries, the
comparisons among means were carried out through the Tukey's HSD farming of mealworms is carried out by family enterprises; in the
test. Netherlands, edible insects belonging to this species can already be
Viable counts of lactic acid bacteria were subjected to χ2 test be- purchased for human consumption in specialized shops (van Huis et al.,
cause of the low frequency of samples with counts ≥ 1 log cfu g− 1 for 2013). Very recently, the potential use of mealworms for the industrial
this microbial group. An overall χ2 test (3 degrees of freedom) was manufacture of minced meat-like products has successfully been ex-
carried out by comparing the frequencies of samples below and above plored (Stoops et al., 2017).
1 log cfu g− 1 across all the four producers, followed by χ2 tests for The ANOVA results from the plate counting data collected from the
specific comparisons. 40 samples of mealworm larvae are reported in Table 2, and the mean
Due to the very low frequency of samples showing viable counts of values of viable counts and multiple comparisons among means are
Enterobacteriaceae ≥ 1 log cfu g− 1, data for this microbiological para- reported in Table 3. As a general trend, viable counts were in the range
meter were not subjected to either ANOVA or χ2 tests. of those reported by other authors for the same microorganisms in heat
The analysis of AR determinants was started by creating a data processed and dried mealworms intended for human consumption
matrix where PCR- and nested PCR-positive and -negative samples were (Garofalo et al., 2017; Grabowski and Klein, 2017; Milanović et al.,
scored as 1 or 0, respectively. A 40 × 40 matrix of Jaccard Similarity 2016; Osimani et al., 2017a). It is noteworthy that in this study, the
indexes was further obtained from the previous data matrix and a samples produced in Thailand were determined to have the lowest
Principal Coordinate Analysis (PCoord) was conducted to study the mean viable counts for all the tested microorganisms; this evidence
grouping pattern of the whole set of 40 samples obtained from four might be likely explained by the well-established tradition of Thailand
different producers (NTSYS - Applied Biostatistics Inc., NY, USA). in the production and marketing of edible insects (Maciel-Vergara and
Additionally, relative percentages of positivity for the 12 AR de- Ros, 2017). Among the tested microorganisms, spore-forming bacteria
terminants were calculated for each producer as the number of PCR- or have been detected in both fresh and processed mealworms (Garofalo
nested PCR-positive samples divided by the total number of samples et al., 2017; Stoops et al., 2016); it has previously been suggested that
assayed (=10). From this 4 × 12 data matrix the Principal Component the guts of these insects naturally contain these microorganisms (Engel
Analysis (PCA) from the Pearson correlation matrix was performed. The and Moran, 2013), though an environmental contamination during
eigenvectors' coefficients of the Principal Components allowed the processing cannot be excluded. Moreover, the overall low occurrence of
quantification of the relative importance of each AR determinant in Enterobacteriaceae, defined as an indicator of processing hygiene
differentiating the four producers. The results were summarized gra- (Brown et al., 2000; Petruzzelli et al., 2016), attests to the appro-
phically by plotting the four producers based on their respective PCA priateness of the heat treatment of the edible insects assayed.
scores (NTSYS - Applied Biostatistics Inc., NY, USA). The overall per- Regarding total mesophilic aerobes, mean counts ranged be-
centages of positivity for the 12 determinants were also calculated as tween < 1 and 6.73 log cfu g− 1. Multiple comparison among

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A. Osimani et al. International Journal of Food Microbiology 263 (2017) 38–46

Table 3
Mean counts ( ± standard deviation) of total mesophilic aerobes, spore-forming bacteria, lactic acid bacteria, and Enterobacteriaceae in samples of edible mealworms from the four
producers located in the Netherlands (NL1-NL10), Thailand (TH1-TH10), Belgium (BL1-BL10) and France (FR1-FR10).

⁎ ⁎
Samples Total mesophilic aerobes Spore-forming bacteria Lactic acid bacteria Enterobacteriaceae

NL1 2.63 ( ± 0.21) a 2.11 ( ± 0.20) a 1.93 ( ± 0.21) <1

NL2 3.10 ( ± 0.05) a 2.30 ( ± 0.14) a 2.99 ( ± 0.06) <1
NL3 3.03 ( ± 0.11) a 2.50 ( ± 0.10) a <1 <1
NL4 3.54 ( ± 0.04) a 2.60 ( ± 0.02) a <1 <1
NL5 3.20 ( ± 0.02) a 2.99 ( ± 0.06) a <1 <1
NL6 3.04 ( ± 0.05) a 3.12 ( ± 0.11) a <1 <1
NL7 3.45 ( ± 0.02) a 2.73 ( ± 0.05) a <1 <1
NL8 3.40 ( ± 0.12) a 2.74 ( ± 0.06) a <1 <1
NL9 3.30 ( ± 0.04) a 2.36 ( ± 0.15) a <1 <1
NL10 2.73 ( ± 0.10) a 2.20 ( ± 0.04) a <1 <1
TH1 0.65 ( ± 1.00) de 0.65 ( ± 0.92) ab <1 <1
TH2 <1 e <1 b <1 <1
TH3 1.70 ( ± 0.10) abcd 0.80 ( ± 1.13) ab <1 <1
TH4 2.24 ( ± 0.30) ab <1 b <1 <1
TH5 2.30 ( ± 0.00) ab 1.00 ( ± 1.40) ab <1 <1
TH6 <1 e <1 b <1 <1
TH7 0.80 ( ± 1.13) cde 1.35 ( ± 1.91) ab <1 <1
TH8 1.24 ( ± 0.30) bcd 1.45 ( ± 0.64) ab <1 <1
TH9 2.40 ( ± 0.10) a 2.62 ( ± 0.05) a 1.54 ( ± 0.09) <1
TH10 1.90 ( ± 0.04) abc <1 b <1 0.50 ( ± 0.61)
BL1 4.30 ( ± 0.03) a 4.01 ( ± 0.05) a 1.72 ( ± 0.17) <1
BL2 4.10 ( ± 0.20) a 3.53 ( ± 0.07) a 2.80 ( ± 0.23) <1
BL3 4.10 ( ± 0.23) a 4.20 ( ± 0.05) a <1 <1
BL4 4.20 ( ± 0.12) a 4.24 ( ± 0.00) a <1 <1
BL5 4.30 ( ± 0.10) a 4.10 ( ± 0.03) a <1 <1
BL6 4.05 ( ± 0.40) a 3.97 ( ± 0.00) a <1 0.52 ( ± 0.71)
BL7 3.83 ( ± 0.05) a 3.70 ( ± 0.07) a <1 <1
BL8 3.96 ( ± 0.08) a 4.20 ( ± 0.05) a <1 <1
BL9 4.02 ( ± 0.00) a 3.50 ( ± 0.10) a <1 <1
BL10 3.70 ( ± 0.02) a 3.99 ( ± 0.02) a 1.60 ( ± 0.00) <1
FR1 4.58 ( ± 0.08) ef 1.69 ( ± 0.12) abc 2.77 ( ± 0.02) <1
FR2 5.77 ( ± 0.04) abcd 2.34 ( ± 0.10) ab 2.50 ( ± 0.02) <1
FR3 5.56 ( ± 0.03) bcde 2.47 ( ± 0.07) ab <1 <1
FR4 6.73 ( ± 0.06) a 2.26 ( ± 0.16) ab 1.35 ( ± 0.50) <1
FR5 3.78 ( ± 0.00) f 2.47 ( ± 0.10) ab 0.50 ( ± 0.70) <1
FR6 5.19 ( ± 0.13) cde 2.74 ( ± 0.12) a 2.45 ( ± 0.21) <1
FR7 4.89 ( ± 0.01) de 2.56 ( ± 0.12) ab <1 <1
FR8 5.98 ( ± 0.00) abc 0.66 ( ± 0.92) bc <1 0.65 ( ± 0.92)
FR9 5.40 ( ± 0.02) bcde 2.73 ( ± 0.06) a 1.77 ( ± 0.10) <1
FR10 6.44 ( ± 0.13) ab <1 c 1.30 ( ± 0.00) <1

Counts are expressed as log colony forming units (cfu) g− 1.

⁎
For total mesophilic aerobes and spore-forming bacteria, within each producer, multiple comparisons among means were carried out through the Tukey's HSD test; within each
producer means followed by different letters are significantly different (P < 0.05).

producers showed that, for this microbial group, the four dealers dif- agreement with the results of previous studies (Garofalo et al., 2017;
fered significantly (Table 2), with those located in France and Thailand Milanović et al., 2016; Osimani et al., 2017b), the nested PCR assays
showing the highest (5.43 log cfu− 1) and the lowest (1.32 log cfu− 1) showed a higher sensitivity (up to 7 orders of magnitude) with respect
counts, respectively. Furthermore, comparisons carried out within each to the corresponding PCR assays, thus allowing the number of samples
producer (Table 3) highlighted the occurrence of a significant variation considered as positive to be increased from 9.4% to 30.4%.
among samples produced in Thailand and France, with viable counts Regarding tetracycline resistance genes, a high prevalence of tet(K)
ranging from < 1.00 to 2.40 log cfu− 1and 3.78 to 6.73 log cfu− 1, re- was noted in all the samples assayed, with percentages of positive PCR
spectively. By contrast, samples from the Netherlands and Belgium results that ranged from 80% (samples from Thailand) to 100% (sam-
were much more homogeneous (Table 3). ples from the Netherlands, Belgium and France). In addition, about half
For spore-forming bacteria, viable counts were between < 1 and of the 40 samples analyzed were positive for both tet(M) and tet(S).
4.24 log cfu g− 1 (Table 3), with samples from Belgium and Thailand Specifically, tet(M) was detected in 100% of the samples produced in
showing the maximum (4.24 log cfu− 1) and minimum (0.65 log cfu) the Netherlands and Belgium whereas no positive samples for this gene
values, respectively (Table 2). Moreover, similarly to what observed for were found in mealworms produced in Thailand and France. The oc-
total mesophilic aerobes, even for spore-forming bacteria Thailand and currence of tet(S) was highest in mealworms from the Netherlands
France were characterized by a significant variation among samples, (100%) and France (70%). Finally, there was an overall low detection
whereas no significant differences were seen among samples produced of tet(O), with this gene occurring only in the 10% of samples produced
in the Netherlands and Belgium (Table 3). in Thailand and France, respectively, and being not detected at all in
Finally, for lactic acid bacteria, viable counts varied between < 1 Belgium and the Netherlands.
and 2.99 log cfu g− 1 (Table 3). The χ2 test showed that the producer Tetracyclines represent one class of antibiotics most commonly used
located in France differed from the other three with a significantly in human and veterinary clinical practice due to their broad spectrum
higher frequency of samples with counts of lactic acid bacteria above of activity. Resistance to these antibiotics is usually conferred by the
1 log cfu g− 1 (χ2(1) = 8,54, P < 0.01). acquisition of a tetracycline resistance gene (tet) encoding for efflux
The results of PCR and nested PCR are reported in Table 4. In pump systems [e.g., tet(K)] or ribosomal protection [e.g., tet(M), tet(O),

41
A. Osimani et al. International Journal of Food Microbiology 263 (2017) 38–46

Table 4
Results of PCR and nested-PCR amplification of AR determinants in samples of ready-to-eat edible mealworms samples from the four producers located in the Netherlands (NL1-NL10),
Thailand (TH1-TH10), Belgium (BL1-BL10) and France (FR1-FR10).

Samples Assays Antibiotic resistance determinants

erm(A) erm(B) erm(C) vanA vanB tet(M) tet(O) tet(S) tet(K) mecA blaZ aac-aph

NL1 PCR − − − − − − − + + − − −
n-PCR − − − − − + − n.d. n.d. + − −
NL2 PCR − − − − − + − + − − − −
n-PCR − + + − − n.d. − n.d. + − − −
NL3 PCR − − − − − + − + + − − −
n-PCR − − + − − n.d. − n.d. n.d. − − +
NL4 PCR − − − − − + − + + − − −
n-PCR − − − − − n.d. − n.d. n.d. − − +
NL5 PCR − − − − − + − + + − − −
n-PCR − − − − − n.d. − n.d. n.d. − − +
NL6 PCR − − − − − − − + + − − −
n-PCR − − − − − + − n.d. n.d. − − −
NL7 PCR − − − − − + − + − − − −
n-PCR − − + − − n.d. − n.d. + − − −
NL8 PCR − − − − − − − + − − − −
n-PCR − + − − − + − n.d. + − − −
NL9 PCR − − − − − − − + − − − −
n-PCR − − + − − + − n.d. + − − +
NL10 PCR − − − − − + − + + − − −
n-PCR − − − − − n.d. − n.d. n.d. − − −
NL % of positivity for each determinant 0 20.0 40.0 0 0 100 0 100 100 10.0 0 40.0

TH1 PCR − − − − − − − − − − − −
n-PCR − + + − − − − − − − − −
TH2 PCR − − − − − − − − − − − −
n-PCR − − − − − − − − + − − −
TH3 PCR − − − − − − − − − − − −
n-PCR − + − − − − − − + − − +
TH4 PCR − − − − − − − − − − − −
n-PCR − + − − − − − − + − − −
TH5 PCR − − − − − − − − − − − −
n-PCR − + + − − − − − + − − −
TH6 PCR − − − − − − − − − − − −
n-PCR − + − − − − + − + + − −
TH7 PCR − − − − − − − − − − − −
n-PCR − + − − − − − − − − − −
TH8 PCR − − − − − − − − − − − −
n-PCR − + − − − − − − + − − +
TH9 PCR − − − − − − − − − − − −
n-PCR − + − − − − − − + − − −
TH10 PCR − − − − − − − + − − − −
n-PCR − + − − − − − n.d. + − − −
TH % of positivity for each determinant 0 90.0 20.0 0 0 0 10.0 10.0 80.0 10.0 0 20.0

BL1 PCR − − − − − − − − − − − −
n-PCR − + − − − + − − + − − −
BL2 PCR − − − − − + − − − − − −
n-PCR − + − − − n.d. − − + − − −
BL3 PCR − − − − − + − − − − − −
n-PCR − − − − − n.d. − − + − − +
BL4 PCR − − − − − − − − − − − −
n-PCR − − − − − + − − + − − +
BL5 PCR − − − − − − − − − − − −
n-PCR − − − − − + − − + − − +
BL6 PCR − − − − − − − − − − − −
n-PCR − − − − − + − − + − − −
BL7 PCR − − − − − + − − − − − −
n-PCR − − − + − n.d. − − + − − −
BL8 PCR − − − − − + − − − − − −
n-PCR − − − − − n.d. − − + − − −
BL9 PCR − − − − − + − + + − − −
n-PCR − − − − − n.d. − n.d. n.d. − − +
BL10 PCR − − − − − − − + − − − −
n-PCR − − − − − + − n.d. + − − −
BL % of positivity for each determinant 0 20.0 0 10.0 0 100 0 20.0 100 0 0 40.0

FR1 PCR − − − − − − − + − − + −
n-PCR + + − + − − − n.d. + − n.d. −
FR2 PCR + − − − − − − + − − − −
n-PCR n.d. + + − − − − n.d. + − − −
FR3 PCR − − − − − − − + − − + −
n-PCR − + + + + − − n.d. + − n.d. −
(continued on next page)

42
A. Osimani et al. International Journal of Food Microbiology 263 (2017) 38–46

Table 4 (continued)

Samples Assays Antibiotic resistance determinants

erm(A) erm(B) erm(C) vanA vanB tet(M) tet(O) tet(S) tet(K) mecA blaZ aac-aph

FR4 PCR + + − − − − − + − − − −
n-PCR n.d. n.d. − + − − − n.d. + − − −
FR5 PCR − + − − − − − + − − − −
n-PCR − n.d. + + − − − n.d. + − − −
FR6 PCR − − − − − − − − − − − −
n-PCR − + + + − − − − + − − −
FR7 PCR − − − − − − − − − − − −
n-PCR − + − + − − − − + − − −
FR8 PCR − − − − − − − − − − − −
n-PCR − + + + − − − − + − + −
FR9 PCR − − − − − − − + − − + −
n-PCR − + + + − − + n.d. + − n.d. −
FR10 PCR − − − − − − − + − − − −
n-PCR − + + + − − − n.d. + − − −
FR % of positivity for each determinant 30.0 100 70.0 90.0 10.0 0 10.0 70.0 100 0 40.0 0

Overall % of positivity for each determinant 7.5 57.5 32.5 25.0 2.5 50.0 5.0 50.0 95.0 5.0 10.0 25.0

PCR: positive after PCR; n-PCR: positive after PCR and nested PCR; n.d. not determined.

tet(S)] (Hwang et al., 2017; Wilcks et al., 2005). respectively, whereas vanB was exclusively found in one sample pro-
The frequencies of the four tet genes observed in this study are duced in France. These findings are particularly interesting since an
consistent with those reported by Milanović et al. (2016) and Osimani increasing emergence of vancomycin-resistant strains of Enterococcus
et al. (2017b) in ready-to-eat edible insects, where tet(K), tet(M) and tet faecium has recently been reported by the ECDC (2016).
(S) prevailed, and only a few samples were positive for tet(O). Tetra- To the best of the authors' knowledge, this report is the first to
cycline resistance genes were also found by Larson et al. (2008) in feed describe the occurrence of resistance genes to vancomycin in edible
mill insects and by Pai (2013) in Gram-positive and -negative bacteria insects; indeed, in analogous studies carried out by Milanović et al.
isolated from cockroaches (Periplaneta americana and Blattella germa- (2016) and Osimani et al. (2017b), neither vanA nor vanB could be
nica). In addition, Levy and Marshall (2013) argued that the honeybee detected using the same PCR-based approach. Moreover, in the study
gut can harbor tetracycline resistance genes as the result of selective carried out by Pai et al. (2005) onto house cockroaches (Periplaneta
pressures exerted by the use of tetracyclines in the honeybee industry. americana and Blattella germanica), no vancomycin resistant gram-po-
Regarding resistance to erythromycin, erm(B) prevailed (57.5%), sitive bacteria were isolated.
being retrieved in 90% and 100% of the samples produced in Thailand Regarding beta-lactamase genes, low occurrences for both blaZ and
and France, respectively. The genes erm(A) and erm(C) were retrieved mecA were seen in the 40 samples assayed. More specifically, no sam-
with lower frequencies, at 7.5% and 32.5% of the total samples, re- ples produced in the Netherlands, Thailand and Belgium that were
spectively. However, 70% of mealworms produced in France were po- positive for blaZ, whereas 40% of the samples produced in France were
sitive for erm(C), which was also detected in samples from the positive for this gene. Similarly, just one sample produced in the
Netherlands and Thailand. No samples positive for erm(C) were found Netherlands and one in Thailand was positive for mecA. Regarding this
in the samples produced in Belgium. Interestingly, all the samples po- latter determinant, the overall low occurrence of beta-lactamase genes
sitive for erm(A) were produced in France. is in agreement with the results of Milanović et al. (2016) and Osimani
Macrolides inhibit the synthesis of bacterial proteins and represent et al. (2017b).
one of the most important classes of antibiotics in the clinical treatment Beta-lactams are one of the first described classes of antibiotics;
of community-acquired pneumonia, sexually transmitted diseases, sal- their anti-bacterial activity is carried out by beta-lactamases, which are
monellosis, and shigellosis. Resistance to these antibiotics is encoded by enzymes capable of hydrolysing the beta-lactam ring (Queener, 1986).
erythromycin ribosomal methyltransferase genes (Fyfe et al., 2016). As reported by the ECDC, EFSA, and European Medicines Agency
In the present study, the detection frequencies of erm genes are (EMA) (2009), methicillin-resistant Staphylococcus aureus and Clos-
comparable to those reported by Milanović et al. (2016) and Osimani tridium difficile can enter the food chain; moreover, strains of Strepto-
et al. (2017b) in various edible insects; indeed, in both studies, erm(A) coccus pneumoniae, S. aureus and Mycobacterium tuberculosis are now
was undetected, whereas erm(B) and erm(C) were present with fre- showing an alarming increase in resistance to beta-lactams, and their
quencies ranging from 16.7 to 45.4% and from 18.2 to 26.7%, re- efficacy is worryingly declining (Fisher and Mobashery, 2016).
spectively. To the authors' knowledge, no other published studies are Finally, regarding the gene aac-aph conferring resistance to ami-
available in the scientific literature to date on the occurrence of ery- noglycosides, 40% of the samples produced in the Netherlands and in
thromycin resistance genes in either edible or non-edible insects. The Belgium and 20% of the samples produced in Thailand were positive for
overall low occurrence of erm genes in the 40 samples of mealworm this determinant. By contrast, no samples positive for aac-aph were
larvae analyzed is in agreement with data reported by the European found in mealworms produced in France. Aminoglycosides are broad-
Food Safety Authority (EFSA) and the European Centre for Disease spectrum antibiotics routinely used against aerobic Gram-negative
Prevention and Control (ECDC) (EFSA and ECDC, 2016). Notwith- bacteria, for their capacity of binding to bacterial ribosomes, and hence
standing, EFSA and ECDC (2016) highlighted that, although resistance of inhibiting the synthesis of bacterial proteins (Mingeot-Leclercq et al.,
to erythromycin is considered as not being subject to transfer between 1999). Resistance to this class of antibiotics can be exerted via N-
different strains of bacteria, horizontal transfer of resistance genes to acetylation, O-nucleotidy-lation, and/or O-phosphorylation inactiva-
this class of antibiotics has been described in Asia, between bacteria tion and efflux pumps (Sheikhalizadeh et al., 2017). To the authors'
ascribed to genus Campylobacter. knowledge, there is a shortage of scientific literature on resistance to
Concerning genes for resistance to vancomycin, vanA was detected aminoglycoside antibiotics in insects. The aac-aph detection frequency
in 90% and 10% of the samples produced in France and Belgium, found in this study was similar to that reported by Osimani et al.

43
A. Osimani et al. International Journal of Food Microbiology 263 (2017) 38–46

0.75

0.50
FR10 FR3
FR5 NL7
FR9 NL3
FR2 NL2 NL9
FR1 NL6
0.25 FR4 NL10
FR8
NL1 NL4
BL10 NL5
PCoord 2

FR6
NL8 BL9
TH10
0.00
TH1
TH5 BL7

FR7
-0.25 TH6 BL6 BL3
BL8 BL4
TH7 TH2 BL5
BL1
TH3 BL2
TH9
-0.50 TH4 TH8

-0.75
-0.75 -0.50 -0.25 0.00 0.25 0.50 0.75
PCoord 1

Fig. 1. Results of principal coordinates analysis of AR determinants showing the distribution of samples based on the first and second principal coordinates.
Letters refer to the samples listed in Table 3.

(2017b) onto grasshoppers. The fact that edible insects can constitute a coli (Wilke et al., 2005); members of both these taxa have already been
reservoir of resistance to aminoglycosides has first been suggested by detected in mealworm larvae by Stoops et al. (2016) and Osimani et al.
Larson et al. (2008), in whose study strains of Enterococcus gallinarum (2017a). Finally, for the genes conferring resistance to aminoglycosides,
isolated from Sitophilus zeamais Motschulsky (maize weevil) and Tro- a correlation with the occurrence of Enterobacteriaceae, such as E. coli
goderma variabile Ballion (warehouse beetle) were resistant to strepto- and Klebsiella spp., recently found in the microbiota of mealworms
mycin and neomycin. As reported by the ECDC (2017), aminoglycoside larvae (Osimani et al., 2017a), can be hypothesized.
resistance, in combination with third-generation cephalosporin and Results of PCoord Analysis (Fig. 1) evidenced a good grouping of
fluoroquinolone resistances, has significantly increased in the EU/EEA samples purchased from the same producer. The first principal co-
(European Economic Area) between 2012 and 2015 for both Escherichia ordinate separated samples produced in France and Thailand, which
coli and Klebsiella pneumoniae, thus representing a threat for patients showed negative scores, from those produced in the Netherlands and
suffering from illnesses caused by these two human pathogens. Belgium, which showed positive scores. The second principal co-
It is acknowledged that genes that confer resistance to a given an- ordinate further distinguished within these two groups, separating the
tibiotic can be specifically associated, although not limited, to a specific samples produced in France and the Netherlands, with positive scores,
bacterial genus or group, as already reported for the erm and tet genes from those produced in Thailand and Belgium, with negative scores.
(Chopra and Roberts, 2001; Leclercq, 2002; Milanović et al., 2017). Overall, PCoord showed a separation of the four producers based on the
To date, tet genes have been associated with numerous microbial distribution of the 12 AR determinants, though a partial overlapping of
genera, including Bacillus, Clostridium, Streptococcus, Staphylococcus, clusters of samples from the same producers was observed. PCA was
Enterobacter, Escherichia, Klebsiella, Lactobacillus, Lactococcus, and further applied in order to identify the relative load of each AR de-
Pseudomonas, with tet+ microorganisms being increasingly isolated terminant in the differentiation among the four producers. Eigenvalues
from various food matrices (Bulajić et al., 2015; Devirgiliis et al., 2013; and eigenvectors are reported in Table S3 (Supplementary material)
Eid et al., 2015; Verraes et al., 2013). Regarding mealworms, recent and graphic of PCA scores in Fig. 2. The producer from France was
studies aimed at identifying the microbiota of both fresh and dried clearly separated from the other three by PC1, based on a relatively
larvae (Garofalo et al., 2017; Osimani et al., 2017a; Stoops et al., 2016) higher frequency of erm(A), erm(B), erm(C), vanA, vanB, tet(O), and
have highlighted the presence of numerous species belonging to the blaZ, which were characterized by highly positive eigenvector coeffi-
above mentioned genera, thus potentially explaining the high occur- cients (close to + 1), and a lower frequency of tet(M) and aac-aph,
rence of tet genes in the analyzed samples. which were by contrast characterized by highly negative eigenvector
Similarly, erm genes have been detected in a number of foodborne coefficients (close to − 1).
microorganisms, including Bacillus, Enterobacter, Lactobacillus, and Concerning PC2, erm(B), tet(M), tet(O), tet(S) and tet(K) dis-
Pseudomonas (Tang et al., 2015; Xing et al., 2014); representatives of criminated the producer from Thailand from the other three producers
these latter genera have previously been retrieved in mealworm larvae on the basis of a lower detection frequency of tet(M) and tet(S) (char-
(Garofalo et al., 2017; Osimani et al., 2017a; Stoops et al., 2016), thus acterized by highly positive eigenvector coefficients) and a higher de-
supporting once again the hypothesis of a contribution of erm+ mi- tection frequency of erm(B) and tet(O) (characterized by highly negative
croorganisms belonging to these genera to the high occurrence of erm eigenvector coefficients). Finally, PC3 separated the producer from the
genes in the analyzed samples. Netherlands from that from Belgium, the latter being characterized by a
Further hypothesis can be formulated to correlate the detection of higher frequency of tet(S) and mecA, both characterized by highly ne-
the remaining AR genes with microbial genera or species previously gative eigenvector coefficients.
detected in mealworms larvae. At this regard, enterococci, which have Overall, both PCoA and PCA could differentiate the four producers
very recently been detected by PCR-DGGE and pyrosequencing in dried from each other; this finding suggests a potential role of geographical
mealworms larvae (Garofalo et al., 2017), are known to contribute to locations on such variability, as the result of the differences potentially
the spread of vancomycin resistance genes in the food environment occurring in clinical practices and antimicrobial usage in the respective
(Van den Braak et al., 1997). Regarding beta-lactamase genes, it has countries of origin. This hypothesis also seems to be supported by the
previously been proved that determinants responsible for resistance to results previously obtained by the same authors on different edible
beta-lactamases can effectively be carried by Pseudomonas spp. and E. insect species (Milanović et al., 2016; Osimani et al., 2017b).

44
A. Osimani et al. International Journal of Food Microbiology 263 (2017) 38–46

FR Fig. 2. Results of Principal Component Analysis of AR de-

terminants showing the variation characterizing the 4 pro-
BL
ducers based on AR determinant frequencies.
Letters refer to the samples listed in Table 3.

PC3

NL
TH

PC
2
P C1

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define a baseline for health risk assessment on edible insects as a food Antimicrobial Resistance. CAC/GL, pp. 77–2011.
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occurrence of foodborne ARs should be considered in the risk analysis sistance genes within the gastrointestinal tract of lesser mealworm larvae, Alphitobius
diaperinus (Coleoptera: Tenebrionidae). Foodborne Pathog. Dis. 6 (7), 907–915.
of food products. Given the growing interest on the use of mealworms Devirgiliis, C., Barile, S., Perozzi, G., 2011. Antibiotic resistance determinants in the in-
as a novel protein source, AR detection frequencies found in the present terplay between food and gut microbiota. Genes Nutr. 6, 3275–3284.
study suggest further insights on the use of antibiotics during rearing of Devirgiliis, C., Zinno, P., Perozzi, G., 2013. Update on antibiotic resistance in foodborne
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prudent use of antibiotics during rearing of edible insects is re- EFSA and EMEA on meticillin resistant Staphylococcus aureus (MRSA) in livestock,
companion animals and foods. EFSA-Q-2009-00612 (EFSA Scientific Report (2009)
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301, 1-10) and EMEA/CVMP/SAGAM/62464/2009. Available online: http://www.
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