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Acinetobacter baumannii is a Gram-negative bacterium that has gained a stronghold inside healthcare settings. Due to the ability of A. baumannii to acquire antibiotic resistance easily, its presence in food products could pose a major threat to the public health. The aim of this study therefore, was to investigate the prevalence of A. baumannii in fresh produce and study their genetic diversity. A total of 234 samples of vegetables and fruits were collected. A. baumannii isolates were identified using CHROMagar and two different PCR assays. Also, the isolates were tested for their ability to resist antibiotics and form biofilms. The genetic diversity of the isolates was determined using multi-locus sequence typing (MLST). Of the 234 samples collected, 10 (6.5%) and 7 (8.75%) A. baumannii isolates were recovered from vegetables and fruits, respectively. Antibiotic susceptibility testing revealed that 4 of these isolates were extensively drug-resistant (XDR). All isolates were able to form biofilms and MLST analysis revealed 6 novel strains. This study demonstrated that fresh produce constitutes a reservoir for A. baumannii , including strong biofilm formers and XDR strains. This represents a significant concern to public health because vegetables and fruits may serve as a vehicle for the spread of A. baumannii and antibiotic resistance into the community and healthcare settings.
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Ababnehetal.
International Journal of Food Contamination (2022) 9:5
https://doi.org/10.1186/s40550-022-00092-7
RESEARCH
Fresh produce asapotential vehicle
fortransmission ofAcinetobacter baumannii
Qutaiba Ababneh*, Ekhlas Al‑Rousan and Ziad Jaradat
Abstract
Acinetobacter baumannii is a Gram‑negative bacterium that has gained a stronghold inside healthcare settings. Due
to the ability of A. baumannii to acquire antibiotic resistance easily, its presence in food products could pose a major
threat to the public health. The aim of this study therefore, was to investigate the prevalence of A. baumannii in fresh
produce and study their genetic diversity. A total of 234 samples of vegetables and fruits were collected. A. bauman-
nii isolates were identified using CHROMagar and two different PCR assays. Also, the isolates were tested for their
ability to resist antibiotics and form biofilms. The genetic diversity of the isolates was determined using multi‑locus
sequence typing (MLST). Of the 234 samples collected, 10 (6.5%) and 7 (8.75%) A. baumannii isolates were recovered
from vegetables and fruits, respectively. Antibiotic susceptibility testing revealed that 4 of these isolates were exten‑
sively drug‑resistant (XDR). All isolates were able to form biofilms and MLST analysis revealed 6 novel strains. This study
demonstrated that fresh produce constitutes a reservoir for A. baumannii, including strong biofilm formers and XDR
strains. This represents a significant concern to public health because vegetables and fruits may serve as a vehicle for
the spread of A. baumannii and antibiotic resistance into the community and healthcare settings.
Keywords: Acinetobacter baumannii, Fresh produce, MLST, Biofilm formation, Antibiotic resistance
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Introduction
Acinetobacter baumannii is a Gram-negative bacterium
that has become an immensely dangerous pathogen
inside healthcare settings due to its ability to resist mul-
tiple groups of antimicrobial agents (Nasr 2020). is
pathogen can cause a wide range of diseases, including
urinary tract infections, skin and soft tissue infections,
bacteremia, pneumonia, osteomyelitis, and meningi-
tis (Williams etal. 2020). Several factors contributed to
the success of A. baumannii as a nosocomial pathogen,
including its capacity to adapt to adverse environmen-
tal conditions, desiccation resistance, antibiotic resist-
ance and genome plasticity. Besides, A. baumannii can
survive exposure to regularly used disinfectants such as
phenols and chlorhexidine, and it can tolerate the dry
environment for months (Gallego 2016).
Although A. baumannii is commonly known as a noso-
comial pathogen, it has also been isolated from diverse
sources such as food, water, soil and animals (Lupo etal.
2014; Rafei et al. 2015; Al Atrouni et al. 2016; Karum-
athil etal. 2016; Carvalheira etal. 2017a; Carvalheira etal.
2017b). e presence of A. baumannii in food is considered
a serious problem, as contamination of the food chain with
this bacterium might enable it to find its way into health-
care settings, and thus exacerbate the burden of nosoco-
mial infections caused by this pathogen (Lupo etal. 2014).
In recent years, consumption of the fresh produce (fruits
and vegetables) has increased due to modernization of the
agriculture methods and the surge in production (Carval-
heira etal. 2017a). Consumption of raw or minimally pro-
cessed fresh produce can serve as a source for the spread
of this pathogen, both in communities and hospital envi-
ronments (Berlau et al. 1999). Vegetables and fruits may
Open Access
International Journal
of Food Contamination
*Correspondence: qoababneh@just.edu.jo
Department of Biotechnology and Genetic Engineering, Faculty of Science
and Arts, Jordan University of Science and Technology, P.O. Box 3030,
Irbid 22110, Jordan
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Page 2 of 9
Ababnehetal. International Journal of Food Contamination (2022) 9:5
acquire A. baumannii while growing in the soil, during har-
vesting, from organic fertilizers, from contaminated irriga-
tion water, as well asduring transportation and handling
(Machado-Moreira etal. 2019). Moreover, vegetables and
fruits have high-water activity, which helps in the growth of
microorganisms, including Acinetobacter species.
Few studies reported the isolation of A. baumannii
from fresh produce. For example, this pathogen have
been isolated from apple, melon, bean, carrot, potato,
and radish (Berlau etal. 1999), while a Japanese group
have isolated it only from leek (Oie etal. 2008). In addi-
tion, two studies reported the contamination of lettuce
samples with A. baumannii (Karumathil etal. 2016; Car-
valheira et al. 2017b). However, none of these studies
attempted to elucidate the link of this pathogen with the
clinical context, as the A. baumannii clonality of strains
investigated was not determined. Information about the
clonality of A. baumannii isolated from fresh produce
will increase our understanding of any potential exchange
of A. baumannii clones between food and healthcare
settings. erefore, this study aimed to investigate the
prevalence clonality, antibiotic resistance and biofilm for-
mation of A. baumannii in fresh produce collected from
retail markets in the city of Irbid, Jordan.
Materials andmethods
Samples collection andisolation ofA. baumannii
A total of 234 samples (154 vegetables and 80 fruits)
were collected between October 2018 and February 2020
from different hypermarkets and retail markets in Jordan
(Table1). A. baumannii was isolated from all samples fol-
lowing the procedure described previously (Rafei et al.
2015). Briefly, all samples were processed within 24 h of
collection in a UV-sterilized laminar flow. Ten grams
from each sample were weighted inside the laminar flow
on a sterile aluminum sheet and suspended in 90 mL ster-
ile distilled water (10% w/v). e suspension was homog-
enized in an orbital shaker water bath for 15 min, then
decanted for 30 min. Five milliliters of the suspension
were added to 20 mL of Dijkshoorn enrichment media
and mixed in an orbital shaker water bath at 150 rpm for
48 h at 37°C (Carvalheira etal. 2017b). CHROMagar Aci-
netobacter plates (CHROMagar, France) were used for
samples culturing and incubated for 24–48 h at 37°C. Red
colonies with white halo were regarded as presumptive A.
baumannii and were selected for further analysis.
Molecular identication ofA. baumannii
A. baumannii was identified at the molecular level by
partial polymerase chain reaction (PCR) amplification
of the hyp (Hamouda 2017) and blaOXA-51 genes (Turton
etal. 2006). In addition, a multiplex PCR assay was used
to differentiate between A. baumannii, A. nosocomialis,
and A. pittii (Chen et al. 2014). Genomic DNA was
extracted using Wizard Genomic DNA Purification Kit
(Promega/USA) as per the manufacturer’s instructions.
All PCR products were purified from agarosegels using
GeneJet Gel Extraction Kit (ermoFisher, USA) and
subjected to DNA sequencing (Macrogen, South Korea).
e obtained DNA sequences were analyzed by BLAST
search. DNA isolated from the reference strain A. bau-
mannii ATCC 19606 was included as a positive control
for all PCR assays.
Antibiotic sensitivity testing
e disk diffusion method was used to perform the anti-
biotic sensitivity testing against the following antibiot-
ics: doripenem (10 μg), imipenem (10 μg), meropenem
(10 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), ceftriax-
one (30 μg), cefepime (30 μg), ceftazidime (30 μg), ami-
kacin (30 μg), tobramycin (10 μg), gentamicin (10 μg),
ampicillin-sulbactam (10/10 μg), tetracycline (30 μg),
trimethoprim-sulfamethoxazole (1.25/23.75 μg), pipera-
cillin (100 μg), and piperacillin-tazobactam (100/10 μg).
All antibiotics were purchased from Oxoid, UK. e
minimal inhibitory concentrations (MICs) of colistin
(Sigma, Germany), polymyxin B (Duchefa Biochemie,
Netherlands) and tigecycline (Cayman, USA) antibi-
otics were determined by the broth micro-dilution
method as described previously (Wiegand etal. 2008).
e Clinical and Laboratory Standards Institute (CLSI)
antibiotic susceptibility breakpoints (CLSI 2018) were
used to classify the isolates into susceptible, interme-
diate, or resistant. e reference strain A. baumannii
ATCC19606 was included in all antibiotic susceptibility
tests as controls.
Biolm formation
Biofilm formation was assayed by the semi-quantitative
method in a sterile 96-well microtiter plates as described
previously (Hu etal. 2016). For each isolate, the biofilm
assay was performed in triplicate per 96-well plate, and
the optical densities (ODs) of 3 independent plates were
compared with the cut-off OD (ODc) to determine the
biofilm phenotype as follows; non-biofilm producer:
OD ODc; weak biofilm producer: ODc < OD 2 × ODc;
moderate biofilm producer: 2 × ODc < OD 4 × ODc; or
strong biofilm producer: OD > 4 × ODc.
Multi‑locus sequence typing (MLST)
MLST was performed on all isolates following the Pas-
teur scheme by PCR amplification of internal fragments
of 7 housekeeping genes (cpn60, fusA, gltA, pyrG, recA,
rplB, and rpoB), and subsequent DNA sequencing of
the PCR amplicons (Macrogen, South Korea). e PCR
conditions for MLST are described on the PubMLST
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Ababnehetal. International Journal of Food Contamination (2022) 9:5
website (https:// pubml st. org/ abaum annii/). Amplifica-
tion reactions for the MLST PCR consisted of 20 ng/μl
of extracted DNA, 0.4 μM of each primer, 1X PCR ready
mix (iNtRON, South Korea). Alleles and sequence types
were identified using the tools of the PubMLST database
(Jolley et al. 2018). goeBURST analysis was performed
using the PHYLOViZ tool(version 2.0) as described pre-
viously (Francisco etal. 2012).
Results
Isolation andidentication ofA. baumannii
e fresh produce samples analyzed in this study
included imported (42/234; 18%) and domestic (192/234;
82%) products. All mango and the majority of the apple
samples (18/19) were imported. Other types of samples
were imported include, carrot (6/10), lemon (1/10), pears
(3/10), grapes (1/11), peach (2/12) and chili green pepper
(1/10). All other fresh produce were grown locally.
CHROMagar was used to identify the presumptive
A. baumannii colonies, which were isolated from 150
(64.1%) samples. Most of the tested cucumber, carrot,
lettuce, arugula, mint, parsley, red radish samples con-
tained presumptive A. baumannii isolates. However, not
all the presumptive isolates were identified by PCR as A.
baumannii, instead several isolates were identified as A.
pittii by the multiplex PCR assay and DNA sequencing
(Table1). A. pittii colonies were detected in 54 (23%) of
samples collected.
Table 1 Prevalence of A. baumannii in the collected fresh produce samples
A: No. of hyp gene positive isolates.
B: No. of A. baumannii conrmed by theMultiplex PCR assay.
C: No. of blaoxa-51 gene positive isolates.
d British cucumber, snake cucumber and Cucumis melo
e Red, yellow and green apples
Sample Source No. of samples with conrmed A.
baumannii / No. of samples collected No. of samples with
presumptive A. baumannii
colonies
Molecular
identication of A.
baumannii by PCR
No. of samples with
conrmed A. pittii
isolates
A B C
Vegetables
Tomato 0/10 4 0 0 0 1
Cucumber varieties d3/12 11 3 3 3 3
Carrot 0/10 10 0 0 0 3
Lettuce 0/10 10 0 0 0 8
Lemon 0/10 4 0 0 0 0
Sweet Green Pepper 0/10 8 0 0 0 0
Sweet Yellow Pepper 1/10 8 0 1 1 2
Sweet Red Pepper 1/10 7 0 1 1 2
Arugula 2/10 10 0 2 2 7
Mint 2/10 10 1 2 2 6
Parsley 0/10 10 0 0 0 8
Red Radish 1/10 9 1 1 1 6
Coriander 0/10 10 0 0 0 6
Chili Green Pepper 0/10 6 0 0 0 0
Chili Red Pepper 0/4 1 0 0 0 0
Cherry Tomato 0/8 1 0 0 0 0
Fruits
Apple varieties e2/19 7 1 2 2 0
Pear 2/10 4 1 2 2 0
Grape 1/11 3 1 1 1 0
Strawberry 1/10 5 0 1 1 0
Peach 0/12 6 0 0 0 2
Guava 1/8 2 0 1 1 0
Mango 0/10 4 0 0 0 0
Total 17/234 150 8 17 17 54
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Ababnehetal. International Journal of Food Contamination (2022) 9:5
Approximately 64% of the samples harbored presump-
tive A. baumannii, but only 17 isolates (7.3%) were recov-
ered from the 234 collected samples. Table1 summarizes
the number of presumptive colonies and the confirmed A.
baumannii isolates for each sample. When tested for the
presence of the hyp-gene by uniplex PCR, more than half
of the multiplex PCR-confirmed A. baumannii isolates
tested positive for hyp gene. However, all the multiplex
PCR positive A. baumannii isolates harbored the blaOXA-51
gene. In themultiplex PCR assay, four genes were ampli-
fied; the recA gene that exists in all Acinetobacter spe-
cies, the gyrB gene thatis present only in A. baumannii
and A. nosocomialis,the internal transcribed spacer (ITS)
region of A. baumannii, and theITS region of A. pittii. It
is important to mention that the multiplex PCR results
were confirmed by DNA sequencing. With respect to the
source of the samples, 4 of the 17 confirmed A. baumannii
isolates were recovered from imported products, while the
remaining isolates were from domestic samples. Isolates
AP4 and AP8 were recovered from red apple samples from
Italy and theUSA, respectively. In addition, we isolated
A. baummannii (GP1) from a green grape sample from
Egypt, and one isolate (PR1) from Spanish pears.
Antibiotic sensitivity testing
e majority of the isolates were sensitive to most of the
tested antibiotics except for ceftriaxone, for which all
theisolates displayed resistance or intermediate pheno-
types. Based on their resistance profiles, the isolates were
grouped into 11 resistance patterns (A to K) (Table2). Six
isolates exhibited the same resistance pattern A, while 2
isolates had the same resistance pattern B. Each of the
remaining 9 isolates had different resistance patterns.
Four isolates recovered from red radish, red apple, green
grape, and guava were classified as XDR, however, all of
these isolateswere sensitive to ampicillin-sulbactam anti-
biotic except the isolate that was recovered from guava.
is isolate was sensitive to trimethoprim-sulfamethoxa-
zole, tobramycin, colistin and polymyxin B, while exhib-
ited resistance to tigecycline. e other 3 XDR isolates
were sensitive to colistin, polymyxin B and tigecycline.
Biolm formation
irteen of the 17 isolates (76.5%) displayed strong ability
to form biofilms invitro. Among the 4 XDR isolates, two
were strong biofilm formers, while the other two isolates
were weak and moderate biofilm formers (Table3). e
isolates from cucumber, arugula,red apple, pears, straw-
berry and sweet pepper were all strong biofilm form-
ers. Two isolates recovered from mint and green grapes
were classified as moderate biofilm, and the weak biofilm
formers were recovered from arugula and guava.
Multi locus sequence typing (MLST)
MLST analysis showed that 11 isolates belonged to six
known sequence types (STs), while the other six isolates
were novel strains (Table 3). Five isolates belonged to
ST40, two isolates had ST2 and four isolates belonged to
ST481, ST602, ST724 and ST897. Two of the XDR iso-
lates (AP8 and GP1) recovered in this study belonged to
ST2, one belonged to ST724 while the XDR isolate from
guava had a new ST. Data from the PubMLST Acineto-
bacter database and the literature indicated that strains
belonging to ST481 and ST897 have been previously
isolated from animal sources. ST40 strains have been
isolated from clinical and food sources, while isolates
Table 2 Antibiotic resistance profiles for the A. baumannii isolates
DOR Doripenem, IPM Imipenem, MEM Meropenem, CIP Ciprooxacin, LEV Levooxacin, CRO Ceftriaxone, FEP Cefepime, CAZ Ceftazidime, AK Amikacin, TOB
Tobramycin, CN Gentamicin, SAM Ampicillin-sulbactam, TE Tetracycline, SXT Trimethoprim-sulfamethoxazole, TZP Piperacillin-tazobactam, PRL, Piperacillin, CT Colistin,
POL B Polymyxin B, TGC Tigecycline, ND Not determined, S Sensitive, R Resistant, I Intermediate
Resistance
pattern No. of
isolates Antibiotic resistance phenotypes
DOR IPM MEM CIP LEV CRO FEP CAZ AK TOB CN SAM TE SXT TZP PRL CT POL B TGC
A 6 S S S S S I S S S S S S S S S S ND ND ND
B 2 R R R R R R R R R R R S R R R R S S S
C 1 R R R R R R R R R R R I R R R R S S S
D 1 R R R R R R R R R S R R R S R R S S R
E 1 S I S S S R S I S S S S S S S S ND ND ND
F 1 S S S S S I S S S S S R S S S S ND ND ND
G 1 S S S S S I S S R S I I S S S S ND ND ND
H 1 S S S R R R I I S S S S S S S S ND ND ND
I 1 S S S S S I S I S S S S S S S I ND ND ND
J 1 S S S S S I S S S S S S S S S I ND ND ND
K 1 S S S S S I S S S S S S S I S S ND ND ND
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Ababnehetal. International Journal of Food Contamination (2022) 9:5
belonging to ST602 have been previously isolated from
environmental and upper respiratory tract samples. e
PubMLST database contains data about only one ST724
strain isolated from blood, whereas hundreds of ST2 iso-
lates have been isolated from clinical, animal and envi-
ronmental sources (Brahmi etal. 2016; Khurshid etal.
2020; Shelenkov etal. 2021).
Six isolates had new allelic profiles and were assigned
the new sequence types ST1854-ST1857 and ST1862–
ST1863 (Table4). e data related to these 6 isolates were
deposited in the PubMLST database. Isolate SKCUC1
harbored a new allele sequence of the rpoB gene,
whichwas assigned the allelenumber 318. e sequence
of the rpoB-318 allele shares 99.78% identity with alleles 5
and 48. e other five isolates carried new allelic combi-
nations. eBURST analysis revealed that ST1854, ST1855,
ST1856 and ST1863 were single locus variants (SLV) for
ST40, ST316, ST1036 and ST610, respectively (Fig. 1).
Also, ST1862 is a double locus variant (DLV) to ST764
and ST1354, while ST1857 is a DLV to ST1228.
Discussion
Fresh fruits and vegetables are an integral part of healthy
and balanced diets; providing us with carbohydrates, fib-
ers, minerals, vitamins and many other micronutrients, as
well as protecting us from many diseases such as obesity,
Table 3 Characteristics of all recovered A. baumannii isolates
Isolate Source Antibiotic Resistance Antibiotic
resistance
pattern
Biolm Formation Sequence Type PubMLST source
CUC6 Cucumber Non‑MDR A Strong 897 Animal
RK1 Arugula Non‑MDR A Weak 40 Sputum, urine, upper respiratory
tract, wound, blood, and food
RD2 Red Radish XDR B Strong 724 Blood
MT1 Mint Non‑MDR E Moderate 40 Same as isolate RK1
SYP1 Sweet Yellow Pepper Non‑MDR A Strong 40 Same as isolate RK1
SRP1 Sweet Red Pepper Non‑MDR G Strong 1854 New Type
SKCUC1 Snake Cucumber Non‑MDR I Strong 1857 New Type
CUCML1 Cucumis Melo Non‑MDR A Strong 1856 New Type
MT13 Mint Non‑MDR K Strong 602 Upper respiratory tract and
environment
RK7 Arugula Non‑MDR A Strong 1862 New Type
AP4 Red Apple Non‑MDR J Strong 481 Animal (Dog mouth)
PR1 Pear Non‑MDR A Strong 40 Same as isolate RK1
STY1 Strawberry Non‑MDR F Strong 40 Same as isolate RK1
AP8 Red Apple XDR B Strong 2 Sputum, urine, upper respiratory
tract, wound, blood, and medical
environment
PR2 Pear Non‑MDR H Strong 1855 New Type
GP1 Green Grape XDR C Moderate 2 Same as isolate AP8
GV1 Guava XDR D Weak 1863 New Type
Table 4 Characteristics of the new STs found in the study
Isolate ID Isolate
PubMLST ID MLST genes Assigned
ST Comment
Cpn60 fusA gltA pyrG recA rplB rpoB
SRP1 6923 69 2 2 2 5 1 14 1854 New allelic combination
PR2 6924 3 8 6 2 4 1 5 1855 New allelic combination
CUCML1 6925 3 16 2 2 5 1 4 1856 New allelic combination
SKCUC1 6926 25 3 2 2 156 4 318 1857 New rpoB allele
RK7 6939 12 3 2 2 4 1 14 1862 New allelic combination
GV1 6940 40 2 7 2 40 4 4 1863 New allelic combination
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Ababnehetal. International Journal of Food Contamination (2022) 9:5
cancer and cardiovascular diseases (Iwu and Okoh 2019).
erefore, the worldwide demand for fresh produce has
been increasing in recent years. At the same time, food-
borne illness and disease outbreaks associated with the
consumption of fresh produce have also increased glob-
ally (Machado-Moreira et al. 2019). Contamination of
fresh produce with different types of foodborne pathogens
has been widely demonstrated. Although not regarded as
a foodborne pathogen, A. baumannii have been isolated
from a variety of foods, such as fish, dairy products, meat
and fresh produce. us, food products contaminated
with A. baumannii could be a potential source of infec-
tion for humans, especially if similar A. baumannii strains
were isolated from food and clinical samples. Currently,
there is still a large gap in ourknowledge regarding the
prevalence of A. baumannii in fresh produce, and whether
the presence of A. baumannii in this type of food might
lead to infections in humans. erefore, the aim of this
study was to investigate the prevalence of A. baumannii in
fresh produce collected from local markets in Jordan, and
to determine pathogen characteristics, including genetic
diversity, antibiotic resistance phenotypes and biofilm for-
mation capability.
Fresh produce is considered one of the major con-
tributors to foodborne illnesses when compared to
other dry and non-fresh food products. e Center for
Disease Control and Prevention (CDC) reported that
contaminated fresh produce is the cause of almost 46%
of foodborne illnesses in the US through the period 1998
to 2008 (Karumathil etal. 2016). Fresh produce can be
contaminated with A. baumannii from the soil in the
field due to the use of natural fertilizers or contaminated
irrigation water. Al Atrouni etal. (2016) isolated A. bau-
mannii from soil samples near agricultural zones, which
might have been irrigated with wastewater or reclaimed
water (Al Atrouni etal. 2016). In addition, agriculture
soil might be contaminated from other sources such as
domestic or wild animal feces that graze in the same agri-
cultural area. Indeed, A. baumannii was isolated from
animal’s fecal samples by several research groups (Beu-
chat 1996; Brandl 2006; Rafei etal. 2015; Al Atrouni etal.
2016; Carvalheira et al. 2017a). Additionally, fresh pro-
duce can be contaminated during the harvesting, han-
dling, or transportation processes, or in retail markets
(Brandl 2006; Carvalheira etal. 2017b).
In the present study, 7.3% of the 243 fresh produce
samples examined were found to be contaminated with
A. baumannii, such as cucumber, mint, arugula, red rad-
ish and peppers, which is a major food safety concern,
since these vegetables are frequently used in preparing
raw salads. Moreover, 4 of the 17 A. baumannii isolates
were recovered from imported products. is finding
requires further investigation to elucidate whether these
strains originated from exporting countries or they are
Fig. 1 Minimum spanning tree of MLST data generated by PHYLOViZ. Circles correspond to STs. The ST chosen for analysis shared at least 5 alleles
with the novel STs detected in this study
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Ababnehetal. International Journal of Food Contamination (2022) 9:5
local strains. Nowadays, international importing allows a
continuous supply of fresh produce throughout the year,
and it has been growing significantly due to globalization
of trade and the increased demand (Carstens etal. 2019).
At the same time, imported fresh produce has been
implicated in several multi-national outbreaks and con-
tributed to the introduction of new types of antimicro-
bial resistant determinants and pathogenic bacteria to
the importing countries (Vital etal. 2017; Carstens etal.
2019). erefore, thecontinuous surveillance of micro-
bial contamination of imported and domestic produce
could be proven critical to prevent and reduce the num-
ber of illnesses caused by these food products.
Ten out of the 16 vegetable types investigated were free
of A. baumannii, such as lettuce and carrots, which is in
agreement with the findings of a study by Karumathil
etal. (2016). In this study, only one A. baumannii isolate
was recoveredfrom 100 lettuce samples and no isolates
were found in thecarrotsamples investigated (Karum-
athil etal. 2016). A common practice among local retail-
ers is to wash carrots and lettuce to remove soil traces, as
well as to keep the fresh look on these vegetables. Also,
retailers tend to remove the outer most leaves of lettuce
to make them more appealing to consumers. ese prac-
tices might explain why we were unable to isolate A. bau-
mannii from the carrot and lettuce samples investigated
in this study. A. baumannii was sporadically isolated
from vegetables and fruits in a number of studies, in con-
trast to manyother studies thatreported the isolation of
this opportunistic pathogen. For example, several studies
reported the detection of A. baumannii in raw vegetable
salads, vegetables and fruits (Berlau etal. 1999; Houang
etal. 2001; Bezanson etal. 2008; Oie etal. 2008; Dahiru
and Enabulele 2015; Karumathil etal. 2016; Carvalheira
et al. 2017b). Furthermore,A. baumannii strains were
rarely isolated from fruits, as only a few studies reported
a low prevalence of this pathogen in fruits. In our
studyhowever, 8.75% of fruit samples harbored A. bau-
mannii, with 7 isolates recovered from 5 types of fruits;
apples, pears, grapes, guava and strawberries. Variations
in the types of samples tested and the detection methods
may explain the differences in the prevalence of A. bau-
mannii between the current study and previous ones. e
aforementioned types of fruits are hand-picked, and typi-
cally are not washed before being sold to consumer, thus
cross-contamination from handlers at different stages of
farming, packaging and retailing may have contributed to
the presence of A. baumannii on such types of fruits.
Besides A. baumannii, 52 A. pittii isolates were recov-
ered from vegetables and 2 isolates from fruits. Previous
studies reported the isolation of this species from fresh
produce (Berlau etal. 1999; Rafei etal. 2015; Carvalheira
et al. 2017a). However, higher prevalence of A. pittii
was observed in the fresh produce samples investigated
in the current study. In addition, A. pittii was recovered
from food sources other than fresh produce, such as
meat, cheese and milk (Rafei etal. 2015; Al Atrouni etal.
2016; Carvalheira etal. 2017a; Cho etal. 2018). In recent
years, multi-drug resistant A. pittii has become dominant
in various countries, causing nosocomial infections at a
high rate, especially in intensive care units (Pailhoriès
etal. 2018). erefore, the presence of A. pittii in fresh
produce is alarming and may lead to the spread of this
emerging pathogen into healthcare settings. Continuous
monitoring with molecular epidemiological techniques is
warranted to reduce the spread of the pathogen into the
healthcare systems.
e majority of the recovered isolates were susceptible
to clinically relevant antibiotics. However, 4 isolates from
red radish, red apple, green grape, and guava displayed
resistance to 16 antibiotics, including carbapenems.
Many of these antibiotics are still among the drugs of
choice to treat A. baumannii infections in humans. e
introduction of these XDR isolates via the food chain is a
public health concern because not only limits the thera-
peutic options available to treat infections caused by such
strains, but it may contribute to transferring antibiotic
resistance determinants to the gut microbiota in humans.
Furthermore, the presence of antibiotic-resistant patho-
gens on fresh produce might contribute to the distribu-
tion of resistance between different strains, species and
even genera. Horizonal gene transfer via mobile genetic
elements such as plasmids, may enhance the rapid spread
of antibiotic resistance determinants among pathogenic
bacteria, and A. baumannii is no exception. erefore,
continuous monitoring of the presence of antibiotic-
resistant bacteria on fresh produce is important for risk
assessment and implementing food safety interventions.
e other isolates that were sensitive to the drugs might
have been originated from the soil and contaminated the
product during harvesting or handling. us, they were
not in contact with humans, and consequently not in
contact with antibiotics.
Biofilms formed on the surface of fresh produce can
cause serious risks for fresh product quality and public
health, as bacterial biofilms may not be easily removed by
simple washing with water (Bae etal. 2014). Also, certain
types of biofilms are resistant to the cleaning and disin-
fection procedure commonly practiced in fresh produce
retail marketsand the food industry (Joseph etal. 2001;
Lapidot etal. 2006). Many foodborne pathogenic bacteria
can form biofilm, such as Listeria monocytogenes, Staph-
ylococcus spp., Clostridium spp., Salmonella enterica,
Bacillus spp., Escherichia coli, Serratia spp., Campylo-
bacter spp. and Pseudomonas spp. (Bai etal. 2021). Fur-
thermore, biofilm formation is considered an important
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 8 of 9
Ababnehetal. International Journal of Food Contamination (2022) 9:5
virulence factor in A. baumannii. To the extent of our
knowledge, this is the first study to investigate the biofilm
formation capacity of A. baumannii isolated from fresh
produce. All isolates investigated in this study were able
to form biofilms, with the majority classified as strong
formers. is suggests that if these isolates spread into
food preparation facilitates, they may form biofilms on
surfaces within these facilities, and thus become a persis-
tent source of contamination in the food chain.
To the best of our knowledge, this is the first study to
determine the clonality of A. baumannii isolated from
fresh produce. e two XDR isolatesbelonging to ST2
were recovered from two fruit samples(GP1 and AP8)
collected from the same hypermarket. It is known that
infections caused by carbapenem-resistant A. bauman-
nii belonging to ST2 are widespreadin many countries,
us, the presence of ST2 strainsin fruits or vegetables
suggests a possible route of transmission that involves
individuals infected with or carriers for this pathogen.
Furthermore, a number of isolates investigated in this
study belonged to ST40, ST2, and ST602. A. baumannii
strains belonging to these STs were previously isolated
in Jordan from clinical samples (Ababneh etal. 2021b)
and intensive care unit environmental surfaces (Abab-
neh etal. 2021a). is suggests a possible transmission of
these isolates from the hospital sewage to the vegetable
and fruit samples through irrigation water, or by cross-
contamination from handling personnel who might be
infected or colonized with A. baumannii. Two isolates
recovered from cucumber (CUC6) and apple (AP4)
samples belonged to ST481 and ST897, respectively. We
found two records in the A. baumannii Pubmlst data-
base for two strains belonging to ST897; one of which
has been isolated from animals. A strain with ST481 have
been also isolated from an animal source (Pailhoriès etal.
2015). e isolation of ST481 and ST897 strains from
animals might suggest a possible route of transmission of
CUC6 and AP4 isolates to fresh produce through the use
of animal manure as natural fertilizer, wildlife animals or
water runoff containing animal feces.
Sixisolates investigated in this studyare novel strains,
as they didn’t belong to any previously known sequence
typesofA. baumannii. Five of theseisolates were non-
MDR and displayed strong ability to form biofilms
in vitro, which suggest that these are environmental
strains that have not been detected in clinical settings
or exposed to antibiotics. New clones of A. baumannii
are frequently introduced into the community and clini-
cal settings. Due to the high plasticity of A. baumannii
genome, these new clones can eventually develop or
acquire antibiotic resistance if gained entry into clini-
cal settings, which may represent an additional concern.
e ubiquitous distribution of A. baumannii in nature
may allow new strains to be introduced through many
routes into the food processing environments with vari-
ous fresh produce typesor raw foodstuff. Furthermore, if
these strains are able to form biofilms, they will become a
recurrent source of contamination, resistant to disinfec-
tion and potential source of human infections. erefore,
uncovering the food reservoirs of A. baumannii and their
transmission routes within the food chain is important
for preventing the transmission of this pathogen.
Conclusions
To conclude, this study demonstrated that fresh produce
constitutes a reservoir for A. baumannii, including strong
biofilm formers and XDR strains. e presence of A. bau-
mannii in fresh produce represents a significant concern
to public health because vegetables and fruits may serve as
a vehicle for A. baumannii, increasing their dissemination
into the community and healthcare settings. erefore,
continuous monitoring and clonal typing of A. baumannii
strains detected outside clinical settings may increase our
understanding of the population evolution of this patho-
gen, and help predict new possible routes of entry into
clinical settings. Unlike animal food products, fresh pro-
duce is generally consumed with no terminal microbial kill
step, thus the potential risk for human exposure to fresh
produce associated pathogens is greater. Fresh produce
retailers, distributors and farmers must ensure that their
products meet all food safety requirements to prevent A.
baumannii and other pathogens from reaching consum-
ers. On the other hand, consumer should also do their part
in protecting themselves by ensuring that their fresh pro-
duce is washed and cooked thoroughly before eating.
Abbreviations
XDR: Extensively Drug‑Resistant; non‑MDR: Non‑Multi‑Drug Resistant; PCR:
Polymerase Chain Reaction; BLAST: Basic Local Alignment Search Tool; CLSI:
Clinical and Laboratory Standards Institute; OD: Optical Density; MLST: Multi‑
locus sequence typing; MIC: Minimal Inhibitory Concentrations; ITS: Internal
Transcribed Spacer; ST: Sequence Type; SLV: Single Locus Variant; DLV: Double
Locus Variant; DOR: Doripenem; IPM: Imipenem; MEM: Meropenem; CIP: Cipro‑
floxacin; LEV: Levofloxacin; CRO: Ceftriaxone; FEP: Cefepime; CAZ: Ceftazidime;
AK: Amikacin; TOB: Tobramycin; CN: Gentamicin; SAM: Ampicillin‑sulbactam;
TE: Tetracycline; SXT: Trimethoprim‑sulfamethoxazole; TZP: Piperacillin‑tazo‑
bactam; PRL: Piperacillin; CT: Colistin; POL B: Polymyxin B; TGC : Tigecycline; S:
Sensitive; R: Resistant; I: Intermediate; ND: Not Determined.
Authors’ contributions
QA: Conceptualization; Funding acquisition; Supervision; Writing ‑ review &
editing. EA: Data curation; Formal analysis; Methodology; Writing ‑ original
draft. ZJ: Supervision, Formal analysis; Writing ‑ review & editing. The author(s)
read and approved the final manuscript.
Funding
This work was funded by deanship of research at Jordan University of Science
and technology (grant no. 20190498).
Availability of data and materials
Not applicable.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 9 of 9
Ababnehetal. International Journal of Food Contamination (2022) 9:5
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
The authors of the confirm that the manuscript has been read and approve
the final article and that there are no other persons who satisfied the criteria
for authorship but are not listed.
Competing interests
None to declare.
Received: 10 February 2022 Accepted: 20 June 2022
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Disease outbreaks caused by the ingestion of contaminated vegetables and fruits pose a significant problem to human health. The sources of contamination of these food products at the preharvest level of agricultural production, most importantly, agricultural soil and irrigation water, serve as potential reservoirs of some clinically significant foodborne pathogenic bacteria. These clinically important bacteria include: Klebsiella spp., Salmonella spp., Citrobacter spp., Shigella spp., Enterobacter spp., Listeria monocytogenes and pathogenic E. coli (and E. coli O157:H7) all of which have the potential to cause disease outbreaks. Most of these pathogens acquire antimicrobial resistance (AR) determinants due to AR selective pressure within the agroecosystem and become resistant against most available treatment options, further aggravating risks to human and environmental health, and food safety. This review critically outlines the following issues with regards to fresh produce; the global burden of fresh produce-related foodborne diseases, contamination between the continuum of farm to table, preharvest transmission routes, AR profiles, and possible interventions to minimize the preharvest contamination of fresh produce. This review reveals that the primary production niches of the agro-ecosystem play a significant role in the transmission of fresh produce associated pathogens as well as their resistant variants, thus detrimental to food safety and public health.
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