International Journal of Food Microbiology 206 (2015) 1–6

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International Journal of Food Microbiology

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

Short communication

Ready-to-eat street-vended food as a potential vehicle of bacterial

pathogens and antimicrobial resistance: An exploratory study in Porto
region, Portugal
Joana Campos a, Joana Gil b, Joana Mourão a, Luísa Peixe a, Patrícia Antunes a,b,⁎
a
UCIBIO/REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira n° 228, 4050-313 Porto, Portugal
b
Faculdade de Ciências da Nutrição e Alimentação, Universidade do Porto, Rua Dr. Roberto Frias, 4200 Porto, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: The ready-to-eat street vending commerce, as street mobile food vendors, has grown exponentially worldwide,
Received 7 October 2014 representing in some countries a significant proportion of food consumed by the urban population. However, the
Received in revised form 7 April 2015 microbiological food safety hazards of mobile vending units in industrialized countries are scarcely evaluated. To
Accepted 8 April 2015
assess the microbiological quality and safety of this type of food and try to achieve the connection of its contam-
Available online 13 April 2015
ination with hygienic conditions of food-handlers, we analyzed hotdogs (n = 10), hamburgers (n = 10) and
Keywords:
hands (n = 9) from ten street-vending trailers in the Porto region. Food and food-handler samples were tested
Street foods for Enterobacteriaceae and coliform counts, Escherichia coli and coagulase-positive staphylococci counts/detection
Mobile food trailers and presence of Salmonella. Aerobic mesophilic counts and detection of Listeria monocytogenes (Pulsed Field
Food-handlers Gel Electrophoresis-PFGE and serotyping) were also tested in food samples. E. coli isolates were confirmed by
Listeria monocytogenes MALDI-TOF and characterized for clonality (phylogenetic groups-PhG, PFGE and Multilocus Sequence Typing),
Escherichia coli antibiotic resistance (disk diffusion, PCR/sequencing) and intestinal pathogenic virulence factors (PCR/sequencing).
Antibiotic resistance All food samples presented poor microbiological quality (100% Enterobacteriaceae and coliforms; 20% E. coli
(4 hamburgers, 4 trailers) and 20% (2 hamburgers/2 hotdogs, 3 trailers) were positive for L. monocytogenes
(2 PFGE-types belonging to serotype 1/2a and 4b). Salmonella and coagulase-positive staphylococci were not
detected. Food-handlers carried Enterobacteriaceae and coliforms (100%), E. coli (11%) and/or coagulase-
positive staphylococci (44%). E. coli was detected in 12 samples (n = 30-food/food-handlers; phylogenetic
groups A0/A1/B1) with 33% resistant to one or more antibiotics. Two multidrug resistant atypical E. coli
pathotype strains (astA-ST165(CC165)/food-handler, eaeA-ST327/food) were detected. Three out of eight
E. coli clonal lineages [ST409/ST976(CC10)/ST297] and the two L. monocytogenes clones were spread in different
samples/trailers, suggesting cross-contamination or a common source of contamination. This exploratory study,
in Porto region, showed ready-to-eat street foods from vending trailers as potential vehicles of clinically relevant
L. monocytogenes serotypes and/or E. coli carrying clinically relevant virulence/antibiotic resistance features, and
food-handlers as a critical risk factor in this expanding food sector.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction WHO, 2010). Ready-to-eat foods, particularly those which are

Street food vending is found worldwide with great diversity namely
due to socio-economic and cultural factors, representing in some
countries a significant proportion of the food consumed by the urban
population (Burt et al., 2003; Lues et al., 2006; Rane, 2011; WHO,
2010). However, the safety of street food has become one of the major
concerns for public health, since the potential for unsafe or unsanitary
food handling by mobile food vendors is substantial (Burt et al., 2003;
⁎ Corresponding author at: Universidade do Porto, Faculdade de Ciências da Nutrição e
Alimentação, Rua Dr. Roberto Frias, 4200 Porto, Portugal. Tel.: + 351 22 5074320;
fax: +351 22 5074329.
E-mail address: patriciaantunes@fcna.up.pt (P. Antunes).
composed of meat/poultry and salads, prepared and/or sold by vendors
in streets have been recognized as potential vehicles of microbial
foodborne bacteria (e.g. Salmonella, Listeria monocytogenes, entero-
pathogenic Escherichia coli) (Cardinale et al., 2005; Cho et al., 2011;
El-Shenawy et al., 2011; WHO, 2010). In fact, street-vended foods are
usually produced in small mobile units (e.g., vans, trailers or carts)
from which food is sold, mostly with inadequate layout and equipment,
frequently associated with poor environmental sanitation, improper
food handling and storage practices, as well as low quality of raw mate-
rials (Burt et al., 2003; Mamun et al., 2013; Manguiat and Fang, 2013;
WHO, 2010). Moreover, a growing food safety hazard is related to the
role of food in human exposure to antimicrobial resistant bacteria,
either zoonotic, commensal or from environmental origin (DANMAP,

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.04.016
0168-1605/© 2015 Elsevier B.V. All rights reserved.
2 J. Campos et al. / International Journal of Food Microbiology 206 (2015) 1–6

2013; EFSA, 2008, 2014). Although food products of animal origin are
considered the main source of antibiotic resistance dissemination,
fresh food of plant origin, such as salads, frequently used in ready-to-
eat street foods/meals, is becoming a current concern (Campos et al.,
2013; EFSA, 2008). Street-vended food reported hazards, including
antibiotic resistant bacteria, have been studied in developing countries,
but are scarce in industrialized countries. The aim of this study was to
assess the microbiological quality and safety of ready-to-eat street-
vended foods and try to achieve the connection of its contamination
with hygienic conditions of food-handlers in the Porto region.
2. Materials and methods
2.1. Sampling plan
The samples were obtained from vendors in a ready-to-eat state
representing the selling conditions of the foods. Two food types, hotdogs
(n = 10) and hamburgers (n = 10), and food-handler (n = 9) samples
were collected from street-vending trailers operating in Porto (n = 8)
and Vila Nova de Gaia (n = 2) (arbitrarily designated I to X) in January
and February of 2013. The hotdog (sausage) or hamburger included also
bread, ham, cheese, chips, salad and sauces, and were collected using sterile
bags at time of purchase. The glove-juice method, which consisted of dip-
ping both hands in a sterile bag containing 100 mL of enrichment medium
Buffered Peptone Water (Courtenay et al., 2005) was performed on food-
handlers who were preparing the sandwiches, in 7 out of 10 vending
trailers. All samples were transported and stored in refrigeration conditions
and analyzed in a maximum period of 24 h.
2.2. Microbiological quality and safety analysis
Twenty-five grams of each food sample was added to a culture
medium/diluent (1:10 and homogenized for 2 min in a Stomacher),
in agreement with standard methods for aerobic mesophilic count (ISO
4833:2013-1 protocol), Enterobacteriaceae (ISO, 21528-2:2004 protocol),
coliforms (AFNOR/NF Bio 12/20-1206.2010 protocol), E. coli (ISO, 16649-
2:2001 and AFNOR/NF Bio 12/20-1206.2010 protocol), coagulase-positive
staphylococci (ISO 6888-1:1999; 2003), L. monocytogenes (ISO, 11290-
1:2004) and Salmonella (ISO 6579:2002). In addition to direct plating,
food samples were also plated for E. coli detection after an enrichment
step (Buffered Peptone water 37 °C/18 h) for further characterization of
isolates obtained in samples with counts b 10/g (limit method detection).
Food samples were classified as satisfactory, borderline and unsatisfactory
according to the national microbiological guidelines for ready-to-eat
foods (Santos et al., 2005; Fig. 1). For L. monocytogenes we used the EU mi-
crobiological criteria (applied when food business operator is not able to
demonstrate that the product will not exceed the limit of 100 CFU/g
throughout the shelf-life) (Commission Regulation (EC) N°, 2073/2005).
Identification of L. monocytogenes isolates was confirmed by API Listeria
(bioMérieux) and clonal relatedness was assessed by Pulsed-Field Gel
Electrophoresis (PFGE) following AscI and ApaI digestion of genomic
DNA according to the standard 1 day protocol of the CDC (PulseNet,
2013). Salmonella serotype Braenderup H9812 restricted with XbaI
was used as reference standard. PFGE patterns were analyzed using
InfoQuest™ FP software v4.5 (BioRad Laboratories). The average from
experiments was used to cluster the similarity matrix determined by
each single enzyme analysis (AscI and ApaI) using the Dice coefficient
with a 1.0% optimization/tolerance and the unweighted pair group meth-
od with arithmetic averages (UPGMA). Isolates with different PFGE-types
were further serotyped by agglutination by the French National Reference
Centre and WHOCC for Listeria (Institut Pasteur, Paris, France). The strains
were serotyped on the basis of somatic and flagellar antigens, according
to Seeliger and Höhne (1979) with the use of commercially available
antisera (Denka Seiken Co., Tokyo, Japan), except for serum IX, and
WHO-CC for Listeria reference antisera (Seeliger and Höhne, 1979).
For the analysis of food-handlers we proceeded to manual shaking of
each sample bag and performed counting (plating 1 mL directly from
suspension) and detection (plating 1 mL after enrichment step) of
Enterobacteriaceae, coliforms, E. coli, and coagulase-positive staphylococci
and detection of Salmonella, using the same protocols described for food
samples. To evaluate the hygienic condition of food-handlers' hands
our criteria were “satisfactory” in the absence of each microbiological
parameter searched for.
2.3. Characterization of E. coli isolates
To determine the potential sources of contamination, we performed
the characterization of diverse E. coli colonies, a broadly used hygiene
and antibiotic resistance indicator bacteria (DANMAP, 2013; EFSA,
2014; HPA, 2009), obtained from the different samples (with and

Fig. 1. Percentage of samples classified as unsatisfactory (“US”), borderline and satisfactory (“S”) for microbiological parameters. Microbiological guidelines for ready-to-eat food (CFU/g):
Aerobic mesophilic count, “US”: ≥105 and “S”: ≤103; Enterobacteriaceae, “US”: N103; Coliforms, “US”: N103; Escherichia coli, “US”: ≥10 and “S”: b10; Coagulase-positive Staphylococcus,
“S”: b102; Salmonella spp., “S”: absence in 25 g (Santos et al., 2005); Listeria monocytogenes, “US”: present in 25 g and “S”: absence in 25 g (Commission Regulation (EC) N°, 2073/
2005). Microbiological criteria for the assessment of hygienic condition of the hands of food handlers, “US”: presence/hand and “S”: absence/hand. I–X: Street-vending units classified
as unsatisfactory for each microbiological parameters (food-handler samples were absent from vending units V, VIII and X).
 J. Campos et al. / International Journal of Food Microbiology 206 (2015) 1–6
without enrichment). E. coli identification was confirmed by MALDI-
TOF MS (Bruker Daltonics). The assignation of E. coli phylogenetic
groups was carried out by the multiplex PCR assay previously described
(Clermont et al., 2000). Antimicrobial susceptibility tests were performed
by the disk diffusion method following Clinical and Laboratory Standards
Institute guidelines (CLSI, 2013) and/or European Committee on Antimicro-
bial Susceptibility Testing (EUCAST, 2014), including E. coli ATCC 25922
as control strain. Susceptibility to amoxicillin—10 μg, ciprofloxacin—5 μg,
chloramphenicol—30 μg, gentamicin—10 μg, kanamycin—30 μg,
nalidixic acid—30 μg, streptomycin—10 μg, sulfamethoxazole—300 μg,
tetracycline—30 μg, and trimethoprim—5 μg was studied. We used the clin-
ical breakpoints defined by CLSI for Enterobacteriaceae (CLSI, 2013) and ep-
idemiological cut-off (ECOFF) values to amoxicillin, ciprofloxacin,
chloramphenicol, gentamicin, nalidixic acid and trimethoprim defined for
E. coli by EUCAST (http://www.eucast.org). Multidrug resistance (MDR)
was considered when the isolates were resistant to two or more antibiotics
of different families. The study of resistance to several extended spectrum
β-lactams (ceftazidime, ceftriaxone, cefotaxime, cefepime, cefoxitin, aztreo-
nam) and the double disk synergy test (DDST) for detection of extended-
spectrum β-lactamases (ESBL) was performed in Mueller-Hinton 2 agar
(biomérieux) in E. coli resistant to amoxicillin (EUCAST, 2014). For detection
of carbapenemase producers, biochemical test Blue-Carba was performed
in all E. coli isolates (Pires et al., 2013).
From each sample (with and without enrichment) we selected E. coli
isolates of different phylogenetic groups or antibiotic resistance pheno-
types for further molecular studies. Clonal relatedness was assessed by
PFGE following XbaI digestion of genomic DNA (Antunes et al., 2006).
Salmonella Braenderup H9812 (CDC) was used as molecular size
marker. E. coli isolates belonging to different PFGE-types were further
characterized by Multilocus Sequence Typing (MLST) according to the
available database (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). Genes
encoding resistance to sulfamethoxazole (sul1, sul2 and sul3), tetracy-
cline [tet(A), tet(B) and tet(G)], chloramphenicol (floR, cmlA and catA),
amoxicillin (blaTEM, blaSHV, and blaCTX-M), streptomycin (aadA and
strA-strB), trimethoprim (dfrA1 and dfrA12) and ciprofloxacin [qnrA,
qnrB, qnrC, qnrD, qnrS, qepA, oqxAB, aac(6′)-Ib-cr,] and mutations in the
quinolone resistance-determining region (QRDR) of gyrA and parC were
searched by PCR (Antunes et al., 2006, 2007, 2011; Kim et al., 2009;
Minarini and Darini, 2012). Detection and characterization of class 1
integrons were performed by PCR and sequencing in E. coli isolates resistant
to sulfamethoxazole. Detection of intestinal pathogenic E. coli virulence
gene markers of Shiga toxin-producing (STEC) (stx1, stx2 and eaeA), entero-
pathogenic (EPEC) (bfpA and eaeA), enterotoxigenic (ETEC) (estA, eltB and
astA), enteroaggregative (EAEC) (aggR, astA and pCVD) and enteroinvasive
(EIEC) (ial) strains were searched by PCR (Fujioka et al., 2013; Nguyen et al.,
2005). Positive and negative controls were included in all PCR reactions.
Plasmid incompatibility group classification was based on rep-PCR typing
method in specific E. coli isolates with integron and/or virulence factors
and its genomic location by S1 nuclease and Iceu-I digestion of total geno-
mic DNA followed by PFGE and further hybridization with specific probes
(intI1, astA, eaeA and rep) (Antunes et al., 2011).
3. Results and discussion
3.1. Microbiological quality and safety assessment of street food and
food-handlers
All ready-to-eat food samples (n = 20) collected from the ten street-
vending trailers presented unsatisfactory microbiological quality for at
least two of the parameters analyzed, due to the presence of N105 CFU/g
aerobic mesophilic microorganisms (n = 18; 90%) and N 103 CFU/g
bioindicators Enterobacteriaceae and coliforms (n = 20; 100%) (Santos
et al., 2005) (Fig. 1). Although those counts could not be related to the
safety of food products, namely those carrying raw salad vegetables
(HPA, 2009), they are indicative of raw material poor quality and han-
dling practices, including hygiene and/or temperature and time control
3
(Cho et al., 2011; Mamun et al., 2013; Manguiat and Fang, 2013). Also,
the occurrence of E. coli exceeding the ≥ 10 CFU/g limit in four (20%)
hamburger samples acquired from four vending trailers (40%) is of
concern, since E. coli is used as indicator for fecal contamination of
ready-to-eat products (Cho et al., 2011; HPA, 2009). Nevertheless,
after enrichment step we detected E. coli isolates in 55% (n = 11) of
both food types studied. This detection rate was higher compared
with other studies involving the same type of street foods, but in differ-
ent continents (Asia and South America) (Cho et al., 2011; Hanashiro
et al., 2005), which enhance the need to improve those food-handlers'
hygiene procedures. Furthermore, all food-handler samples (n = 9)
of the seven vending trailers analyzed presented poor personal
hygiene, with the presence of different bioindicators. High levels of
Enterobacteriaceae (between 1.7 × 103 CFU/hand and N7.5 × 103 CFU/
hand) and coliforms (between 1.0 × 103 CFU/hand and N 7.5 × 103 CFU/
hand) were detected in five and four food-handlers, respectively, with
additional presence of E. coli in one of them. Despite that all food sam-
ples were classified as satisfactory for coagulase-positive staphylococci
(n = 19, b10 CFU/g; n = 1, 6.0 × 10 CFU/g), those bacteria were detected
(between b 50 CFU/hand and N 7.5 × 103 CFU/hand) in four food-
handlers' samples (44%) from three vending trailers (Fig. 1). Street
food-handlers in Brazil (Bezerra et al., 2010) and South Africa (Lues
et al., 2006) also showed high levels of bioindicators (coliforms and
E. coli) and coagulase-positive staphylococci, suggesting food-handlers
as a source of food contamination. In fact, in our study, food-handlers
who showed worse hand hygiene conditions, including high levels of
bioindicators (trailers III, IV, VI, VII), presence of E. coli (trailer VI) or
coagulase-positive staphylococci (trailer III, VII and IX) were related
with food samples showing poor microbiological quality [presence or
high levels of E. coli (III, VI, IX—both food-types); L. monocytogenes
(III—hamburger; IV—hotdog)], highlighting the importance of appropri-
ate hygiene procedures to food safety. Moreover, as noted in other stud-
ies (Aluko et al., 2014; Liu et al., 2014; Burt et al., 2003) inappropriate
behaviors (e.g. hand washing neglected during food preparation,
presence of visibly dirty hands, presence of jewelry) were observed as
well during this study, indicating that these street food-handlers are
unaware of proper food handling practices.
Salmonella was not detected in any sample (food and food-handlers)
(Fig. 1), as also reported in street foods from Brazil and Korea (Bezerra
et al., 2010; Cho et al., 2011), but contrasting with studies from less-
developed countries (Africa and Asia) where most of street foods had
a poor microbiological quality, including the presence of pathogenic
bacteria (Cardinale et al., 2005; El-Shenawy et al., 2011; Lues et al.,
2006; Manguiat and Fang, 2013; Modarressi and Thong, 2010;
Tabashsum et al., 2013; Zaghloul et al., 2014). Nevertheless, in our
study, L. monocytogenes was detected in four foods (20%) of both
types obtained from three vending trailers (Fig. 1). Those isolates were
associated with two AscI/ApaI PFGE profiles (Fig. 2), belonging to two dis-
tinct serotypes (1/2a and 4b), with one spread in both food types from
the same trailer (X-1/2a) and other in different trailers (III, IV-4b), suggest-
ing cross-contamination or a common source of contamination. Few studies
from Portugal also observed a high frequency of L. monocytogenes contam-
ination in ready-to-eat products (Barbosa et al., 2013; Felicio et al., 2007;
Henriques et al., 2014), mostly associated with the clinically relevant sero-
types 1/2a and 4b, emphasizing the need to determine the potential sources
or vehicles of food contamination. In Europe, sporadic listeriosis cases and/
or outbreaks are often associated with serotypes 1/2a and 4b and a diverse
range of ready-to-eat food products (EFSA, 2013; EFSA, 2015), highlighting
the need for improvements in cleaning and hygiene practices and temper-
ature control in the preparation and storage of such foods.
3.2. Characterization of the bioindicator E. coli
3.2.1. Clonal relatedness
E. coli isolates (n = 30/23 obtained after enrichment) from different
phylogenetic groups (A0/n = 9, food/food-handlers; A1/n = 8, food;
4 J. Campos et al. / International Journal of Food Microbiology 206 (2015) 1–6

Combined PFGE_AscI-ApaI PFGE_AscI PFGE_ApaI

70 80 90
100 Isolate Food type Vending unit

L7 Hamburger III
L8 Hot dog IV
L28 Hot dog X
L29 Hamburger X
S. Braenderup H9812 (reference standard)

Fig. 2. AscI and ApaI PFGE restriction patterns found among Listeria monocytogenes isolates from ready-to-eat food types. PFGE patterns were analyzed by InfoQuest FP version 5.4 software
(BioRad Laboratories). The average from experiments was used to cluster the similarity matrix determined by each single enzyme analysis (AscI and ApaI) using the Dice coefficient
(1.0% band tolerance; 1.0% optimization) and the unweighted pair group method with arithmetic averages (UPGMA). Salmonella serotype Braenderup reference standard (H9812)
restricted with XbaI.

B1/n = 13, food) were recovered from 41% (n = 12) of the samples
studied (n = 6, hamburgers; n = 5, hotdogs; n = 1, food-handler; 6
vending trailers). Recent studies have also been increasingly characteriz-
ing A and B1 E. coli strains from different types of food samples (Campos
et al., 2013; Koo et al., 2012; Rúgeles et al., 2010) and/or relating those
with intestinal or extra-intestinal infections (Rodrigues et al., 2013;
Rodríguez-Baño et al., 2012; Rúgeles et al., 2010; Valverde et al., 2009),
which suggests their potential to spread to humans via food chain (Koo
et al., 2012; Manges and Johnson, 2012). Nevertheless, the frequent
identification of A and B1 E. coli phylogenetic groups in the commensal
flora of humans and animals (Koo et al., 2012; Rodríguez-Baño et al.,
2012) also suggests the possible involvement of food-handlers
(e.g. poor personal hygiene) in the contamination of street-vended foods.
From each sample, E. coli isolates belonging to different phylogenetic
groups or antibiotic resistance phenotypes were selected for further
clonal studies. The 14 isolates selected were associated with eight
PFGE-types (phylogenetic groups A0 = 3, A1 = 1 and B1 = 4) belong-
ing to eight sequence types [ST165 (CC165), ST976 (CC10), ST4127
(CC156), ST327, ST297, ST409, ST2077 and ST3168] (Table 1). Most of
those were already associated with E. coli human intestinal [ST165
(CC165), ST327] or extra-intestinal infections [ST976 (CC10), ST297,
ST409] (Naseer et al., 2009; http://mlst.warwick.ac.uk/mlst/dbs/Ecoli)
and carried clinically relevant antibiotic resistance genes. Three clonal lin-
eages were spread in different samples from the same trailer and/or dif-
ferent trailers (Table 1), suggesting cross-contamination or a common
source of contamination. Interestingly, one of those belongs to a world-
wide disseminated clonal complex A1/CC10 (Manges and Johnson, 2012).
3.2.2. Antimicrobial susceptibility and virulence genes
Thirty three percent (n = 10/30) of the isolates were clinically resis-
tant to one or more antibiotics, as has been frequently shown for clinical
isolates (Johnson et al., 2007; Rodrigues et al., 2013; Rodríguez-Baño
et al., 2012), even though carbapenemase or ESBL producers and
plasmid-mediated quinolone resistance mechanisms were not detected.
Isolates which were clinically susceptible to antibiotics presented inhibi-
tion zone diameters above the ECOFFs. The presence of E. coli resistant
isolates was observed in five samples (n = 2, hamburgers; n = 2,
hotdogs; n = 1, food-handler) acquired from 4 out of the 10 vending

Table 1
Characterization of selected Escherichia coli isolates by sample type.

Type/ending unita Sample PhGb PFGE typec MLSTd Antibiotic resistance Location Chr or PL (Kb; Inc)e

Phenotypef/genotype Classe 1 integron—bp Virulence genes

(gene cassetes)

Hot dogs
I C2 A0 A0-1 ST3168 (−) (−)
III C6 A0 A0-2 ST409 (−) (−)
VI C15 A1 A1-1 ST976(CC10) (−) (−)
IX C24 A1 A1-1 (−) (−)
A1 A1-1 NAL (−)
X C28 B1 B1-1 ST4127(CC156) AML,CIP, CLO, KAN, NAL, STR, SUL, TET, TRI/blaTEM, catA, 3000 bp(aacA4-catB3-dfrA1)/
catB3, aadA, strA-strB, sul1-sul2, tetB, dfrA1, aacA4 PL(~146; F)

Hamburgers
I C1 B1 B1-2 ST327 AML, CLO, STR, SUL, TET/blaTEM, floR, strA-strB, sul2, tetA (-) eaeA/Chr
III C7g A0 A0-2 (−) (−)
VI C16 A1 A1-1 (−) (−)
VIII C23g A1 A1-1 (−) (−)
IX C25g B1 B1-3 ST297 (−) (−)
X C29g B1 B1-4 ST2077 TET/tetA (−)
B1 B1-3 (−) (−)

Food handlers
VI C20g A0 A0-3 ST165(CC165) AML, TET/blaTEM, tetA (−) astA/PL (~100; F)
a
Vending units are identified from I to X.
b
PhG, phylogenetic groups.
c
PFGE types in bold are those shared by different samples.
d
ST, sequence type and CC, clonal complex, as identified by Multilocus Sequence Typing.
e
Chromosomal (Chr) and/or plasmid (PL) location of class 1integrons, astA and eaeA genes assessed by hybridization of I-CeuI/S1-digested genomic DNA using int1, astA, eaeA and rep
probes.
f
AML, amoxicillin; CIP, ciprofloxacin; CLO, chloramphenicol; KAN, kanamycin; NAL, nalidixic acid; STR, streptomycin; SUL, sulfamethoxazole; TET, tetracycline; TRI, trimethoprim; (−),
isolates susceptible to all antibiotics tested.
g
Samples classified as unsatisfactory for E. coli quality control parameter (≥10 CFU/g).
 J. Campos et al. / International Journal of Food Microbiology 206 (2015) 1–6
trailers studied. A multiplicity of antibiotic resistant phenotypes was
detected, mostly to tetracycline (n = 9; 30%), ampicillin (n = 7; 23%),
streptomycin (n = 6; 20%), sulfametoxazole (n = 6; 20%), chloram-
phenicol (n = 6; 20%), nalidixic acid (n = 5; 17%), ciprofloxacin (n =
4; 13%) trimethoprim (n = 4; 13%) or kanamycin (n = 3; 10%). Street-
vended foods as vehicles of antibiotic resistant bacteria are rarely studied
(Haryani et al., 2007; Thong and Modarressi, 2011), including in E. coli
(Manguiat and Fang, 2013; Tabashsum et al., 2013) and this study, as
far as we know, is one of the first reports in an industrialized/European
country, highlighting the presence of these bacteria in this food type or
food-handlers. Resistance rates from this study are lower than those
described for other sources (e.g. humans, animals and meat thereof)
(DANMAP, 2013; EFSA, 2014; Johnson et al., 2007), including in
Portuguese ready-to-eat salads (Campos et al., 2013), but comparative
analysis should be taken cautiously because of differences in the study
design (e.g. enrichment steps with antibiotic selective plates).
MDR profiles were observed in 3 clones [ST165 (CC165), ST4127
(CC156), ST327] recovered from food/food-handler samples of three
vending trailers (Table 1). Of note, ciprofloxacin resistance was associated
with a particular strain ST4127, belonging to the B1/CC156 clonal lineage
previously described as carbapenemase or ESBL producer and associated
with sporadic human infections (Nicolas-Chanoine et al., 2013; Seiffert
et al., 2013). The presence of double mutations in gyrA (Ser83-Leu,
Asp87-Asn) and parC (Ser80-Ile, Glu84-Ala) genes was detected,
which are the most commonly associated with clinical E. coli isolates re-
sistant to fluoroquinolones (Bansal and Tandon, 2011; Minarini and
Darini, 2012). Remarkably, this strain also carried other antibiotic resis-
tance genes [blaTEM, catA, strA-strB, sul2 and tet(B)] and an unusual
integron (intI1-aacA4-catB3-dfrA1-sul1-qacEΔ1), mainly reported in
Asian Gram negative isolates from clinical and non-human sources
(Stokes and Gillings, 2011; Xu et al., 2009), located on widespread IncFII
plasmid (Table 1). In fact, IncF plasmids have been described in different
Enterobacteriaceae and associated with high-risk clones and/or clinically
relevant acquired genes (e.g. blaKPC, blaCTX-M, and qnr genes), from
human and animal sources (Carattoli, 2013), enhancing the possibility
of horizontal transfer to human pathogens.
Additionally, two MDR atypical E. coli pathotype strains, belonging to
STs previously associated with intestinal infections, were found in food
and food-handler samples. The food isolate carrying eaeA gene in the
chromosome lacks the other EPEC (bfpA) or EHEC (stx1, stx2) associated
genes. These strains frequently designated as atypical EPEC (Paton and
Paton, 2002; Fujioka et al., 2013) have been increasingly associated
with infections in industrialized countries (Hernandes et al., 2009).
Also, the food-handler isolate carrying astA gene, in a virulence-
antibiotic resistance plasmid IncFII (Villa et al., 2010), was not positive
for the other genes commonly associated with EAEC (aggR and pCVD)
or ETEC (estA and eltB) strains (Paton and Paton, 2002; Fujioka et al.,
2013).
4. Conclusions
This exploratory study, in one region of a developed country,
demonstrates that ready-to-eat street foods from vending trailers are
potential vehicles of clinically relevant L. monocytogenes serotypes
and/or E. coli carrying intestinal pathogenic virulence factors or clinical-
ly relevant antibiotic resistance genes, which might constitute a hazard
to human health. Moreover, we demonstrate that food-handlers'
hygiene status is contributing to the poor microbiological quality and
safety of these products. These data highlight the need for better
street-food vendor education and/or certification and reinforce the
need for appropriate authority supervision or regulatory oversight to
ensure that consumers are enjoying a safe and wholesome product.
Our data also justify the need for additional studies of ready-to-eat
street-vended foods and their potential risk for foodborne illness in
this emerging food sector.
5
Acknowledgments
We are deeply grateful to Nazaré Pestana for critical review of this
paper. We would also like to thank Dr. Alexandre Leclercq (French
National Reference Centre and WHOCC for Listeria, Institut Pasteur,
Paris, France) for the L. monocytogenes serotyping and Dr. Jorge Machado
(Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal) that
gently provided Escherichia coli carrying intestinal pathogenic virulence
factors. This work received financial support from Master in Food Service
Management (Faculdade de Ciências da Nutrição e Alimentação,
Universidade do Porto) and the European Union (FEDER funds through
the Operational Programme for Competitiveness Factors—COMPETE)
and National Funds (FCT, Foundation for Science and Technology)
through project Pest-C/EQB/LA0006/2013. Joana Campos (FRH/BD/
93091/2013) and Joana Mourão (SFRH/BD/77518/2011) were supported
by Ph.D. fellowships from Fundação para a Ciência e a Tecnologia. To all
financing sources the authors are greatly indebted.
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