Contaminação micriobiologica

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Food Control
journal homepage: www.elsevier.com/locate/foodcont
Microbiological contamination of reusable plastic bags for food
transportation
J. Barbosaa, H. Albanoa, C.P. Silvaa,b, P. Teixeiraa,∗
aUniversidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina, Laboratório Associado, Escola Superior de Biotecnologia, Rua Arquiteto Lobão Vital
172, 4200-374, Porto, Portugal
b Colégio Internato dos Carvalhos, Rua do Padrão, 83, Carvalhos, 4415-284, Pedroso, Portugal
A R T I C L E I N F O
Keywords:
Domestic environment
Foodborne pathogens
Reusable plastic bag
Cross-contamination
Antibiotic resistance
A B S T R A C T
Nowadays, with so many concerns for the environment, the use of reusable plastic bags is becoming routine,
instead of the use of polluting single-use plastic bags. However, this is controversial in terms of food safety, since
consumers transport many different foods, which could contaminate their bags and pose a risk to their health
due to cross-contamination.
This study aimed to detect or enumerate several indicators/pathogens from 30 used reusable plastic (poly-
propylene) bags and, to evaluate their antibiotic resistance profiles after identification by 16s rRNA of each
isolated microorganism. Several genera of Enterobacteriaceae, coagulase-negative staphylococci and also Listeria
monocytogenes were found in the reusable plastic bags analyzed. In general, high percentages of antibiotics
resistance were found, highlighting the elevated occurrence of multi-resistant isolates of coagulase-negative
staphylococci and Enterobacteriaceae. This study demonstrates the level and variety of microbial contamination
of some used reusable plastic bags. No correlation was found between microbial levels and the visual appearance
of each bag demonstrating that appearance is not a reliable datum about the bag contamination. We believe that
this study could help the competent authorities taking measures to alert consumers to good food safety practices,
not only in their kitchens, but also in the bags that carry their food.
1. Introduction
Reusable plastic bags for transport of groceries from the store to the
consumer's home have become popular in recent years (Williams,
Gerba, Maxwell, & Si, 2011). In Portugal, this trend became more no-
ticeable from February 2015, since plastic bags began to be taxed
(Martinho, Balaia, & Pires, 2017). This additional cost resulted in an
increase in the use of ‘bags for life’, i.e., resistant reusable plastic bags.
However, “reusable” does not mean “clean”. Reusing plastic bags is
beneficial to the environment, but as already stated “the public should
be mindful of the ability of bacteria to contaminate and survive for long
periods of time. Bacteria can easily transfer from different types of
reusable bags to the hand and back again” (Hilton, 2015). In the U.S.,
reusable grocery bags are considered a new cross-contamination vehicle
that has the potential to pose a significant risk of bacterial cross-con-
tamination (Byrd-Bredbenner, Berning, Martin-Biggers, & Quick, 2013).
Another important issue is that using the same bag for different
purposes increases the risk of contaminating the bag with a whole host
of bacteria. Understanding the importance of using different bags for
different purposes is an important topic for consumers. However, it was
reported that one in three consumers used these bags for more than just
groceries, such as gym bags, toy bags, among other uses and that 75%
of consumers use these same bags for carrying raw meat and other foods
(Williams et al., 2011). In addition, the same authors stated that reu-
sable bags, if not properly washed, could play a role in the cross-con-
tamination of foods. Contaminated reusable grocery bags could pose a
foodborne illness risk - an outbreak of norovirus in a girls' soccer team
was traced to a contaminated reusable grocery bag (Repp & Keene,
2012).
Considering that there are few studies regarding the microbial
contamination of these plastic bags, the objective of this study was to
evaluate the potential for reusable bags being contaminated. For that,
several indicators/pathogens were detected or enumerated, subjected
to 16S rRNA identification and their antibiotic resistance profiles as-
sessed.
https://doi.org/10.1016/j.foodcont.2018.12.041
Received 25 October 2018; Received in revised form 27 December 2018; Accepted 28 December 2018
∗ Corresponding author.
E-mail address: pcteixeira@porto.ucp.pt (P. Teixeira).
Food Control 99 (2019) 158–163
Available online 31 December 2018
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2. Materials and methods
2.1. Sampling
This study was carried out in the Metropolitan Area of Porto,
Portugal, from October to December, 2015. Thirty used and two unused
(control) reusable plastic bags were sampled. These bags were con-
stituted by 100% polypropylene (type of reusable bags sold in most
supermarkets), varying only in size (information about sampling area is
presented in Table 1). Each of the 30 used bags belonged to a different
consumer. All consumers reported that they had no criterion in the type
of food transported, i.e., the analyzed bags transported all types of food,
raw or processed. However, with the exception of some vegetables, all
raw food (meat and fish) was inside individual packages (plastic bags or
polystyrene trays with a plastic film cover). Before sampling all the bags
were visually inspected by the same laboratory technician and classified
as “very little use” or “extended use” and “apparently clean” or “dirty”.
Samples were collected using one cotton swab moistened with sterile
quarter strength Ringer's solution (Lab M, Bury, United Kingdom),
which was scrubbed over all the interior area and re-suspended in 10ml
of Buffered Peptone Water (BPW, Merck, Darmstadt, Germany).
All samples were transported to the laboratory in a refrigerated box
and analyzed as soon as they arrived (within 24 h).
2.2. Microbiological analyses
Appropriate decimal dilutions were prepared in sterile Ringer's so-
lution for microbial enumeration according to ISO Standards: total vi-
able aerobic microorganisms at 30 °C on plate count agar (PCA,
Pronadisa, Madrid, Spain) (ISO 4833-1: 2013) and enterococci on bile
esculin azide agar (BEAA, Biokar Diagnostics, Beauvais, France)
(Ferreira et al., 2006), both incubated at 30 °C for 72 h and
Enterobacteriaceae on violet red bile dextrose agar (VRBD, Merck) (ISO
21528-1: 2000) incubated at 37 °C for 48 h.
Detection of Listeria spp. was performed in half Fraser broth
(Merck), incubated at 30 °C for 24 h, followed by all confirmatory tests
(ISO 11290-1: 1996). Detection of coagulase-positive Staphylococcus
was performed according to a Portuguese Standard (NP 2260: 1986) in
which 1ml of each sample in BPW solution (Sampling section) was sown
in simple Chapman broth (tryptone 5 gl-1; meat extract 6 gl-1; protease
peptone 5 gl-1; NaCl 75 gl-1; lactose 7.5 gl-1; agar 0.5 gl-1) and incubated
at 37 °C during 24 and 48 h. Cultures were then transferred to Baird-
Parker Agar with egg yolk tellurite (BPA, Biokar Diagnostics) and plates
incubated for 24–48 h at 37 °C; characteristic colonies were confirmed
by coagulase test with rabbit plasma (bioMérieux, Marcy l’Etoile,
France). Coliforms at 30 °C and Escherichia coli were detected according
to NP 2164: 1983 and NP 2308: 1986, respectively. After incubation in
simple lactose broth (Lab M) at 30 °C during 48 h, coliforms at 30 °C
were detected by growth and gas production in brilliant green broth
(Oxoid, Basingstoke, United Kingdom) incubated at 30 °C for 48 h, and
E. coli was detected also by growth and gas production in brilliant green
broth and by indole production on peptone water, both incubated at
44.5 °C for 48 h.
Enumeration of total microorganisms at 30 °C, Enterobacteriaceae
and enterococci were also performed for two unused plastic bags im-
mediately after being purchased (control bags).
2.3. Origin of isolates
Colonies (about 10%) of each selective culture media were ran-
domly selected from plates having between 15 and 150 colonies
(Tabasco, Paarup, Janer, Peláez, & Requena, 2007).
Table 1
Enumeration (log CFU/bag) and detection (presence or absence/bag) data of several microorganisms for 30 used reusable plastic bags studied.
Sample Area
(cm2)
Enumeration (log CFU/bag) Detection (presence or absence/bag)
Total microorganisms
at 30 °C
Enterobacteriaceae Enterococci Coliforms
30 °C
E. coli Staphylococcus
coagulase -
Staphylococcus
coagulase +
Listeria spp. Listeria
monocytogenes
1 4838 4.8 < 1.0 2.7 – – + – – –
2 4672 3.4 3.4 2.0 – – + – + –
3 5424 6.3 4.7 2.8 + – + – – –
4 4736 3.5 < 1.0 2.0 – – + – – –
5 4928 3.9 < 1.0 2.5 – – + – – –
6 4440 3.5 3.9 5.7 + – + – – –
7 4636 3.6 2.9 2.9 – – + – – –
8 4988 3.9 < 1.0 < 2.0 – – – – – –
9 5031 4.3 3.9 < 2.0 + – + – – –
10 3298 2.8 < 1.0 < 2.0 – – – – – –
11 6078 2.5 < 1.0 < 2.0 – – + – – –
12 5891 3.4 < 1.0 2.0 – – + – – –
13 6078 5.9 5.2 5.1 – – + – – –
14 6120 3.2 3.5 < 2.0 + – + – – –
15 5990 3.2 3.0 2.3 – – + – – –
16 4890 7.3 4.5 5.2 + – + – – –
17 4890 2.4 < 1.0 < 2.0 – – + – – –
18 4890 3.7 2.8 < 2.0 – – + – – –
19 4890 2.4 < 1.0 < 2.0 – – – – – –
20 4890 3.5 < 1.0 < 2.0 – – – – + +
21 5301 2.4 2.8 < 2.0 – – + – – –
22 5226 1.9 < 1.0 < 2.0 – – + – – –
23 4538 4.9 < 1.0 3.7 + – + – – –
24 4950 3.0 3.0 < 2.0 – – + – – –
25 4928 2.4 < 1.0 < 2.0 – – + – – –
26 4992 2.0 < 1.0 < 2.0 – – + – – –
27 4736 2.5 < 1.0 < 2.0 – – + – – –
28 5159 2.2 < 1.0 < 2.0 – – – – – –
29 5226 2.5 < 1.0 < 2.0 – – + – – –
30 4890 3.9 2.7 < 2.0 – – + – – –
Legend: Presence (+) or absence (−)/bag.
J. Barbosa et al. Food Control 99 (2019) 158–163
159
2.4. LAB identification by 16S rRNA sequencing
Deoxyribonucleic acid (DNA) of the isolates was extracted ac-
cording to the protocol for total DNA purification from Gram-positive
bacteria of the GRS genomic DNA kit – Bacteria -#GK07.0100 (Grisp,
Porto, Portugal).
PCR amplification of the 16S rRNA gene fragments was performed
using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (
5′-GGTTACCTTGTTACGACTT-3′) (Lane, 1991) as described by Vaz-
Moreira et al. (2009). The 16S rRNA gene nucleotide sequences were
used to query the EzBioCloud library (Yoon et al., 2017).
2.5. Antibiotic susceptibility testing
The classification of each isolate in terms of their antibiotic sus-
ceptibility (sensitive, intermediate or resistant) was achieved according
to the Clinical and Laboratory Standards Institute (CLSI, 2012). Listeria
spp. isolate was classified as described by Barbosa et al. (2013).
Antibiotics were chosen for each group of isolates according to their
diverse representation of different classes of antimicrobial agents. The
minimum inhibitory concentrations (MIC; μg/ml) were determined by
ε-test for trimethoprim/sulphamethoxazole (SXT, AB Biodisk, Solna,
Sweden) and by the agar dilution method for thirteen antibiotics. Each
test was carried out on Muller-Hinton Agar (MHA, bioMérieux) with
cations adjusted for penicillin G (Sigma, Steinheim, Germany) and
ampicillin (Fluka, Steinheim, Germany) and on MHA for vancomycin
(Fluka), oxacillin, ceftazidime, chloramphenicol, nalidixic acid, ni-
trofurantoin (Sigma), ciprofloxacin, erythromycin, gentamicin, tetra-
cycline and rifampicin (all kindly supplied by the company Labesfal,
Portugal).
Each experiment was performed in duplicate and all isolates were
grown on plates of MHA and MHA with cations adjusted with no added
antibiotics as negative controls. The quality control strains Enterococcus
faecalis ATCC 29212, Escherichia coli ATCC 25922 and Staphylococcus
aureus ATCC 25923 were used to monitor the accuracy of MICs (CLSI,
2012). Plates were incubated at 37 °C for 24 h.
Isolates exhibiting resistance to, at least, two of the antimicrobial
agents of different classes were considered to be multi-resistant strains.
2.5.1. Determination of mecA gene for staphylocci isolates
According to CLSI (2012), “oxacillin interpretative criteria may
overcall resistance for some coagulase-negative staphylococci, because
some non-S. epidermidis strains for which the oxacillin MICs are
0.5–2 μg/ml lack mecA”. In this sense, all coagulase-negative staphy-
lococci isolates with oxacillin MICs of 0.5–2 μg/mL were tested for the
presence of mecA gene (CLSI, 2012). All the procedures were performed
according to Castro, Santos, Meireles, Silva, and Teixeira (2016).
2.6. Statistical analysis
Statistical analysis was performed with the IBM SPSS Statistics, 24
(IBM Corporation, USA). Differences between the visual appearance of
the plastic bags (“very little use” or “extended use” and “apparently
clean” or “dirty”) and the results of enumeration obtained were com-
pared using the Student t-test. The mean difference was considered
significant at the 0.05 level.
3. Results and discussion
Results for enumeration and detection of different microorganisms
for 30 used reusable plastic bags studied are presented in Table 1.
Counts of total viable microorganisms, Enterobacteriaceae and en-
terococci were below the detection limit of the enumeration technique
for the two unused reusable plastic bags used as control (data not
shown).
Only two samples showed high counts for total viable
microorganisms: sample 3 with 6.3 log CFU/bag and sample 16 with
7.3 log CFU/bag. For most of the bags, counts of Enterobacteriaceae
were below the detection limit of the enumeration technique.
Nevertheless, counts between 3.0 and 5.0 log CFU/bag were found in a
small number of samples. Also enterococci were detected on 26 samples
at levels close to or below the detection limit of the enumeration
technique (2.0 log CFU/bag) and only 4 samples had higher numbers
(samples 6, 13 and 16 with values of ∼5.0 log CFU/bag and sample 23
with 3.0 log CFU/bag).
It is important to highlight that all the inner surface of each bag was
sampled, even areas which probably came into contact with food in-
frequently, in particular the upper areas in the larger bags. So, although
results are reported per bag, these contaminants are probably con-
centrated in zones such as the bottom of the bags where the probability
of cross-contamination to foods is higher.
Each bag was classified taking into account its visual appearance at
the time of sample collection (data not shown). No significant relation
(P > 0.05) was found between the microbial load and the visual ap-
pearance of each bag, i.e. with “very little use” or “extended use” and
“apparently clean” or “dirty”.
No Staphylococcus coagulase positive were detected in any plastic
bags. However, Staphylococcus coagulase negative were detected in 25
of the 30 bags analyzed.
Although none of the samples revealed the presence of Escherichia
coli, coliforms were detected in five bags (3, 9, 14, 16 and 23).
Listeria spp. was detected in two bags and in one of them Listeria
monocytogenes was present.
Our results are in accordance with some of the few studies on the
subject. Williams et al. (2011) found high numbers of bacteria (in-
cluding fecal coliforms) in every reusable bag collected from consumers
outside a grocery store. Summerbell (2009) tested 49 “used” reusable
shopping bags and found that the majority had some bacterial counts,
30% elevated bacterial counts and 12% unacceptable coliform counts.
Interesting to note that Summerbell (2009) and Williams et al. (2011)
showed that no bacteria were found in single use plastic carryout bags
or new reusable bags. Bags containing coliform bacteria could indicate
that they were contaminated by raw meats or other uncooked food
products; their presence demonstrates that bags become
contaminated
and that foodborne pathogens could exist on the bags (van Leeuwen,
2013). In fact, the major problem associated with the presence of
foodborne pathogens in reusable bags is the increased risk of bacterial
cross-contamination. Already in 1997, Bradford, Humphrey, & Lappin-
Scott investigated the ability of two strains of Salmonella Enteritidis PT4
to cross-contaminate from inoculated egg droplets on surfaces onto
melon or beef. The authors found that cross-contamination in each
portion of food occurred in 1 s, when placed on the surfaces where the
egg drops were wet, and up to 1min when the egg droplets were dry
(Bradford, Humphrey, & Lappin-Scott, 1997). Also Buchholz, Davidson,
Marks, Todd, and Ryser (2012) demonstrated the occurrence of cross-
contamination of E. coli O157:H7, even in very low levels, between
fresh-cut leafy greens. The authors proved that E. coli O157:H7 from
one contaminated batch of leafy greens could easily be spread to sub-
sequent batches of uncontaminated product in a processing facility
(Buchholz et al., 2012). Despite the steel stainless be of a different
material from the reusable bags, the study of Kusumaningrum, Riboldi,
Hazeleger, and Beumer (2003) is no less important. The authors studied
the transference of Salmonella Enteritidis, Staphylococcus aureus and
Campylobacter jejuni, at different initial levels, from kitchen sponges to
stainless steel surfaces and from these to slices of cucumber and roasted
chicken fillet. Cross-contamination of all microorganisms occurred from
surfaces to food, with or without pressure applied, with transference
rates varying from 50% to more than 100% for transmission to cu-
cumber slices, and from 25% to 100% for transmission to roasted
chicken fillet slices (Kusumaningrum et al., 2003).
Microorganisms obtained from the enumeration and/or detection
(Table 1) were isolated and identified by 16s rRNA sequence
J. Barbosa et al. Food Control 99 (2019) 158–163
160
(Supplementary Table S1).
Isolates of Listeria spp. detected were identified as L. innocua in
plastic bag 2 and L. monocytogenes in plastic bag 20. The presence of the
foodborne pathogen L. monocytogenes highlights the risk of cross-con-
tamination, since this pathogen is normally found in ready-to-eat, fresh
and non-processed food (Ferreira et al., 2006; Henriques & Fraqueza,
2017; Maćkiw et al., 2016).
Most of the 73 Enterobacteriaceae isolates were identified as Pantoea
spp. (Table S1). The bacterial genus Pantoea comprises many versatile
species that have been isolated from a multitude of environments, such
as aquatic and terrestrial environments, as well as in association with
insects, animals and humans (Walterson & Stavrinides, 2015). All other
genera found, such as Citrobacter spp., or Escherichia spp., are ubiqui-
tously distributed in nature and have been isolated from food, water,
and other environmental sources and in human clinical samples
(Anahory, Darbas, Ongaro, Jean-Pierre, & Mion, 1998; Anuradha, 2014;
Chart, 2012; Hoffmann et al., 2005; Leal-Negredo, Castelló-Abieta,
Leiva, & Fernández, 2017; Wang et al., 2016). Many members of the
Enterobacteriaceae family are responsible for spoilage of a variety of
foods including fruits and vegetables, meats, poultry, eggs, milk and
dairy products, as well as fish and other seafood (Baylis, Uyttendaele,
Joosten, & Davies, 2011). Although there is a need to cook these raw
foodstuffs thoroughly to avoid food poisoning outbreaks (Hennekinne,
Herbin, Firmesse, & Auvray, 2015), the same does not happen with
ready-to-eat products that are cross-contaminated with these products,
which will not undergo any treatment before being ingested.
Between 31 isolates grown on bile esculin azide agar, and firstly
mentioned as enterococci by esculin hydrolysis (Table 1), only 2 iso-
lates were identified by 16s rRNA sequence as Enterococcus gallinarum.
The remaining 29 isolates were identified as belonging to Staphylo-
coccus spp. and other genera of lactic acid bacteria, such as Mar-
inilactibacillus piezotolerans and Aerococcus urinaeequi. Marinilactibacillus
piezotolerans was firstly isolated from deep sub-sea floor sediment by
Toffin et al. (2005), but other species from this genera have been found
in cheeses and spoiled dry cured-hams (Ishikawa et al., 2007; Rastelli,
Giraffa, Carminati, Parolari, & Barbuti, 2005). Aerococcus urinaeequi are
subsequently found in hospital environments and meat-curing brines
and are very similar to enterococci (Rasmussen, 2016). The presence of
the other organisms (lactic acid bacteria and staphylococci) is also not
surprising, since some of these have been found in different foods,
especially in fermented products of meat origin and cheeses (Afzal
et al., 2010; Barbosa, Ferreira, & Teixeira, 2009; Fijałkowski, Peitler, &
Karakulska, 2016; Pesavento, Calonico, Ducci, Magnanini, & Lo Nostro,
2014).
The majority of the 32 isolates of Staphylococcus coagulase negative
were identified as S. epidermidis (15). Most of the species identified have
already been isolated from different ready-to-eat foods such as cheeses,
cured meats, sausages and other fermented food products or smoked
fish (Chajęcka-Wierzchowska, Zadernowska, Nalepa, Sierpińska, &
Łaniewska-Trokenheim, 2015; Fijałkowski et al., 2016; Mainar et al.,
2016; Place, Hiestand, Burri, & Teuber, 2002; Rodrigues et al., 2017).
Since these bags are often reused, and potentially used for multiple
purposes, the possibility of contamination by several food products as
well as the consumer's hands exists and could explain the presence of
some and varied staphylococci (Williams et al., 2011). Rusin, Maxwell,
and Gerba (2002) studied the transference ability of Micrococcus luteus,
Serratia rubidea and phage PRD-1 from fomites (initial inoculum of
approximately 108 CFU/ml or PFU/ml) to hands and the subsequent
transference from the fingertip (initial inoculum of approximately
106 CFU/ml or PFU/ml) to the lip. Although the authors had verified
the highest transference rates from non-porous and hard surfaces, the
numbers of bacteria transferred to the hands after handling porous
surfaces were still very elevated (> 106 cells). From the fingertip to the
lip, the authors observed a similar transference to the one that occurred
from hard surfaces to hands (Rusin et al., 2002).
The percentage of isolates (belonging to each group of bacteria) that
were sensitive, intermediate and resistant to each tested antibiotics is
shown in Table 2. Also distribution of percentage and number of each
species resistant and intermediate resistant to different antibiotics are
presented (Supplementary Table S2).
Of the two isolates of Listeria spp. found, L. innocua (bag 2) was
intermediate resistant to ciprofloxacin and SXT and L. monocytogenes
(bag 20) was only resistant to erythromycin. Apart from this resistance,
the pathogenic strain L. monocytogenes was sensitive to all antibiotics
commonly used as first and second-choice therapy to treat listeriosis, as
has also been observed by others (Maćkiw et al., 2016).
None of the isolates belonging to Enterobacteriaceae family were
resistant to gentamicin and ciprofloxacin. Resistances to nalidixic acid
(bag 20), ceftazidime (bags 6, 18 and 30) and tetracycline (bag 16)
were only observed for a small number of isolates. On the other hand,
38.4% and 12.3% of Enterobacteriaceae isolates were resistant (bags 2,
3, 6, 7, 9, 13, 15, 16, 18 and 23) and intermediate resistant (bags 2, 3, 9,
13, 20 and 24) to nitrofurantoin, respectively. Twenty three out of 73
isolates were multi-resistant (Table S2) and belong to samples 2, 3, 6, 7,
9, 16, 18, 20 and 24. In general, the percentage of Enterobacteriaceae
isolates resistant to the antibiotics tested was low, but this should not be
undervalued, due to the ability of some Enterobacteriaceae to acquire
antibiotic resistances as well as virulence factors (Baylis et al., 2011).
Only resistances to erythromycin, tetracycline, nitrofurantoin and
rifampicin were found for lactic
acid bacteria isolates. Erythromycin
was the antibiotic with the highest number of resistant isolates (50%
Table 2
Percentage of sensitive, intermediate and resistant isolates to each tested an-
tibiotics.
Sensitive
isolates
(n)
Intermediate
isolates (n)
Resistant
isolates
(n)
Listeria group
(n= 2)
Ampicillin 100.0 (2) 0.0 (0) 0.0 (0)
Penicillin 100.0 (2) 0.0 (0) 0.0 (0)
Vancomycin 100.0 (2) 0.0 (0) 0.0 (0)
Gentamycin 100.0 (2) 0.0 (0) 0.0 (0)
Erithromycin 50.0 (1) 0.0 (0) 50.0 (1)
Tetracycline 100.0 (2) 0.0 (0) 0.0 (0)
Ciprofloxacin 50.0 (1) 50.0 (1) 0.0 (0)
Nitrofurantoin 100.0 (2) 0.0 (0) 0.0 (0)
Rifampicin 100.0 (2) 0.0 (0) 0.0 (0)
Chloramphenicol 100.0 (2) 0.0 (0) 0.0 (0)
SXT 50.0 (1) 50.0 (1) 0.0 (0)
Enterobacteriaceae
group (n=73)
Ampicillin 69.9 (51) 13.7 (10) 16.4 (12)
Ceftazidime 94.5 (69) 5.5 (4) 0.0 (0)
Gentamicin 100.0
(73)
0.0 (0) 0.0 (0)
Tetracycline 93.2 (68) 6.8 (5) 0.0 (0)
Ciprofloxacin 100.0
(73)
0.0 (0) 0.0 (0)
Nitrofurantoin 49.3 (36) 12.3 (9) 38.4 (28)
Chloramphenicol 76.7 (56) 17.8 (13) 5.5 (4)
Nalidixic acid 98.6 (72) 0.0 (0) 1.4 (1)
Staphylococcus
group (n=47)
Ampicillin 46.8 (22) 0.0 (0) 53.2 (25)
Penicillin 42.6 (20) 0.0 (0) 57.4 (27)
Oxacillin 61.7 (29) 0.0 (0) 38.3 (18)
Ceftazidime 55.3 (26) 6.4 (3) 38.3 (18)
Vancomycin 100.0 (0) 0.0 (0) 0.0 (0)
Gentamicin 100.0 (0) 0.0 (0) 0.0 (0)
Erithromycin 36.2 (17) 34.0 (16) 29.8 (14)
Tetracycline 85.1 (40) 0.0 (0) 14.9 (7)
Ciprofloxacin 97.9 (46) 2.1 (1) 0.0 (0)
Lactic acid bacteria
group (n=16)
Ampicillin 100 (16) 0.0 (0) 0.0 (0)
Penicillin 100 (16) 0.0 (0) 0.0 (0)
Vancomycin 100 (16) 0.0 (0) 0.0 (0)
Erithromycin 37.5 (6) 12.5 (2) 50.0 (8)
Tetracycline 93.8 (15) 0.0 (0) 6.2 (1)
Ciprofloxacin 100 (16) 0.0 (0) 0.0 (0)
Nitrofurantoin 50.0 (8) 18.8 (3) 31.2 (4)
Rifampicin 75.0 (12) 0.0 (0) 25.0 (4)
Chloramphenicol 100 (16) 0.0 (0) 0.0 (0)
J. Barbosa et al. Food Control 99 (2019) 158–163
161
resistant – bags 13 and 15 - and 12.5% intermediate resistant – bags 3,
5, 6, 12 and 13), followed by nitrofurantoin (31.2% resistant and 18.8%
intermediate resistant – all from bag 13), rifampicin (25% resistant;
bags 3 and 13) and, finally, tetracycline (6.2% resistant; bags 1, 13 and
16). Also multi-resistances were found, being all multi-resistant isolates
from sample 13. Marinilactibacillus piezotolerans isolates were sensitive
to all antibiotics tested, but on the other hand, three isolates of
Aerococcus urinaeequi and all Enterococcus gallinarum (2) were multi-
resistant (Table S2). As previously described, aerococci share some
features of antibiotic resistance with enterococci (reviewed by
Rasmussen, 2016). Antibiotic resistance and/or multi-resistance of En-
terococcus spp. isolated from different processed and ready-to-eat foods
are well described (Barbosa et al., 2009; Pesavento et al., 2014). The
ability of sensitive enterococci to acquire antibiotic resistances is a
cause of concern, combined with the common presence of virulence
factors in a high number of strains (Barbosa, Gibbs, & Teixeira, 2010).
The highest percentages of isolates resistant to the different anti-
biotics tested were found for isolates belonging to the genus
Staphylococcus. More than 50% of isolates were resistant to ampicillin
and penicillin and, of those, 21% (10 isolates from bags 1, 3, 4, 5, 12,
13, 14, 15, 23 and 24) were simultaneously resistant to ampicillin,
penicillin and oxacillin. All resistant isolates with oxacillin MICs of
0.5–2 μg/mL were tested for the presence of mecA gene. None of the
isolates harbored the mecA gene (data not shown), indicating that none
of these multi-resistant isolates were classified as resistant to methi-
cillin. Also the number of isolates resistant to ceftazidime (38.3% re-
sistant – bags 1–7, 11–16 and 23–25 - and 6.4% intermediate resistant –
bags 1, 6 and 15) and erythromycin (29.8% resistant – bags 3, 12, 14,
18, 22, 23, 25–27, 29 and 30 - and 34.0% intermediate resistant – bags
1, 3–6, 11–13, 15, 17, 21, 24 and 27) was elevated. None of the isolates
were resistant to vancomycin or gentamicin. However, only 10 isolates
from 47 were not multi-resistant (Table S2; from bags 1, 3, 7, 9, 12 and
23). Other authors reported high percentages of antibiotic resistant and
multi-resistant coagulase-negative staphylococci isolated from different
foods (Fijałkowski et al., 2016; Nunes, Aguila, & Paschoalin, 2015) and
despite that coagulase negative staphylococci are not classical food
poisoning bacteria, their ability to spread antibiotic resistances to
others of the same genus, as Staphylococcus aureus, or even to other
pathogens, is significantly relevant.
In summary, the use of reusable plastic bags for different purposes
and the transport of different foods, carrying different microorganisms,
can pose a problem of contamination, especially, cross-contamination.
Using separate bags for different classes of products, as meats, fresh
fruits and vegetables, and ready-to-eat foods, as well as their frequent
washing could be a starting point to reduce cross-contamination and it
is already recommended by Government Agency of Food Safety from
USA (Gieraltowski, 2012).
4. Conclusions
In this study, we showed that the 30 analyzed reusable plastic bags
were contaminated with different microorganisms, including patho-
gens. Among them, we also found several antibiotic resistant isolates,
including multi-resistances.
It was demonstrated that appearance is not a reliable datum about
the bag contamination. Microbiological evaluation of the efficacy of
simple washing/sanitising procedures applied in the domestic en-
vironment could be an interesting study to perform with contaminated
reusable plastic bags.
We believe that this study clearly demonstrates the need of educa-
tion campaigns to alert the public about the misuse of their reusable
plastic bags and how this may affect their health. The competent au-
thorities should contribute to tentatively minimize this problem and
one simple measure can start with printing instructions on reusable
bags, alerting to the need for washing and separation of raw and ready-
to-eat foods.
Declarations of interest
None.
Acknowledgments
This work was scientifically supported by National Funds from the
Fundação para a Ciência e a Tecnologia (FCT, Portugal) through project
UID/Multi/50016/2013 and through project “Biological tools for
adding and defending value in key agro-food chains (bio – n2 – value)”,
nº NORTE-01-0145-FEDER-000030, funded by Fundo Europeu de
Desenvolvimento Regional (FEDER, Portugal), under Programa
Operacional Regional do Norte - Norte2020”. Financial support for
author J. Barbosa was provided by a post-doctoral fellowship SFRH/
BPD/113303/2015 (FCT, Portugal).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodcont.2018.12.041.
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	Microbiological contamination of reusable plastic bags for food transportation
	Introduction
	Materials and methods
	Sampling
	Microbiological analyses
	Origin of isolates
	LAB identification by 16S rRNA sequencing
	Antibiotic susceptibility testing
	Determination of mecA gene for staphylocci isolates
	Statistical analysis
	Results and discussion
	Conclusions
	Declarations of interest
	Acknowledgments
	Supplementary data
	References