International Journal of Food Microbiology 318 (2020) 108478

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International Journal of Food Microbiology

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

High prevalence of mcr-1 encoding colistin resistance and first identification T

of blaCTX-M-55 in ESBL/CMY-2-producing Escherichia coli isolated from
chicken faeces and retail meat in Tunisia
Bilel Hassena, Mohamed Salah Abbassia,b, Laura Ruiz-Ripad, Olouwafemi M. Mamad,
⁎
Abdennaceur Hassenc, Carmen Torresd, , Salah Hammamie
a
Université de Tunis El Manar, Institut de la Recherche Vétérinaire de Tunisie, 20 rue Jebel Lakhdhar, Bab Saadoun, Tunis 1006, Tunisie
b
Université de Tunis El Manar, Faculté de Médecine de Tunis, Laboratoire de résistance aux antibiotiques LR99ES09, Tunisie
c
Laboratoire de Traitement et de Valorisation des Rejets Hydriques, Centre des Recherches et des Technologies des Eaux (CERTE), Technopole Borj-Cédria, BP 273, 8020
Soliman, Tunisie
d
Departamento de Agricultura y Alimentación, Universidad de La Rioja, 26006 Logroño, Spain
e
Université la Manouba, IRESA, École Nationale de Médecine Vétérinaire de Sidi Thabet, Sidi Thabet 2020, Sidi Thabet, Ariana, Tunisie.

A R T I C LE I N FO A B S T R A C T

Keywords: Avian industries have been reported as an important contributor in the worldwide spread of antibiotic resistance
Escherichia coli owing to some particular practices especially the overuse of antibiotics. Thus in this study, we aimed to char-
Poultry acterize extended-spectrum-beta-lactamase (ESBL) and acquired-AmpC-beta-lactamase (aAmpC)-producing
ESBL/CMY-2 Escherichia coli isolates from chicken faeces and raw meat in Tunisia. During the year 2018, 286 faecal chicken
mcr-1
swabs and 47 raw chicken meat samples were collected and processed to recover cefotaxime-resistant E. coli.
blaCTX-M-55
Antimicrobial susceptibility was performed by disk-diffusion and/or broth-microdilution. blaTEM, blaSHV, blaCTX-
M, and blaCMY genes were investigated by PCR/sequencing. Genes encoding resistance to colistin (mcr-1 to mcr-
8), tetracycline (tetA/tetB), sulfonamide (sul1/sul3), and chloramphenicol (cmlA), were analysed by PCR. Class 1
integrons were investigated by PCR/sequencing. Phylogenetic groups of all isolates were determined. PFGE and
MLST were performed for representative isolates. PCR-based replicon typing was performed in mcr1-harbouring
isolates. Cefotaxime-resistant E. coli was detected in 22.4% (64/286) and 63.8% (30/47) of faeces and meat
samples, respectively. Ninety isolates were ESBL-producers and harboured the genes: blaCTX-M-1 +/− blaTEM-1
(n = 65), blaCTX-M-55 +/− blaTEM-1 (n = 21), blaCTX-M-14 (n = 1), and blaSHV-12 (n = 3). The blaCMY-2 gene was
detected in four ESBL-negative isolates. Isolates belonged to phylogroups D (50%), A (36.2%), B1 (9.6%), and B2
(4.3%). Fifty-four were colistin-resistant and 52 carried the mcr-1 gene. The tetA, sul1/sul3 and cmlA genes were
detected among resistant isolates and 76 harboured class 1 integrons. MLST analysis revealed 13 sequence types
(STs). The isolates were classified into 28 PFGE types. The IncP, IncFIB, and IncI1 replicons were detected among
mcr-1-positive strains. We report a high frequency of ESBL-producers and colistin-resistant E. coli in chicken and
derived food and the detection for the first time of blaCTX-M-55 in poultry in Tunisia.

1. Introduction aminoglycosides, and thus have prompted the reassessment of colistin

Infections caused by multidrug-resistant Gram-negative bacteria,
are a severe risk for public health and are responsible for outbreaks
worldwide (Cerceo et al., 2016). Extended-spectrum-betalactamase
(ESBL)-producing Enterobacteriaceae have been isolated from various
sources and ecological niches such as humans, livestock, various animal
food and the environment (Paterson, 2006). Furthermore, ESBL-pro-
ducing Enterobacteriaceae are often resistant to fluoroquinolones and
as a critically important antimicrobial for human therapy (Kempf et al.,
2016). For decades, colistin has been used in curative treatments and
considered extensively a prophylaxis drug in veterinary medicine (Skov
and Monnet, 2016). The extensive use of colistin in animals may result
in a highly selective pressure in the veterinary environment leading to
the selection and the spread of colistin-resistant pathogens. Until re-
cently, colistin resistance mechanisms were considered limited to
chromosomal mutations in pmrA/B, phoP/Q, and mgrB genes (Zaman

⁎
Corresponding author at: Departamento de Agricultura y Alimentación, Universidad de La Rioja, Madre de Dios, 53, 26006 Logroño, Spain.
E-mail address: carmen.torres@unirioja.es (C. Torres).

https://doi.org/10.1016/j.ijfoodmicro.2019.108478
Received 11 July 2019; Received in revised form 21 October 2019; Accepted 10 December 2019
Available online 16 December 2019
0168-1605/ © 2019 Elsevier B.V. All rights reserved.
B. Hassen, et al.
et al., 2018). However, an acquired colistin-resistance gene mcr-1 has
been reported in Escherichia coli and Klebsiella pneumoniae in China, and
thereafter rapidly detected in large genera of enterobacteria (Cao et al.,
2018; Grami et al., 2016; Tong et al., 2018). To date, nine mcr variants
have been identified in various species in humans and animals (Carroll
et al., 2019). Among food-producing animals, a multitude of in-
vestigations has demonstrated a notable prevalence of colistin re-
sistance in poultry. Many reports have highlighted the genetic linkage
of ESBL or acquired-AmpC betalactamase (aApmC) encoding genes in E.
coli isolates and the spread of specific clones or plasmids of specific Inc-
types (Carattoli et al., 2018; Maamar et al., 2018). Consequently, the
main aim of this study was to characterize ESBL-/aAmpC producing E.
coli isolates from poultry faeces and poultry meat and to identify genes
encoding colistin resistance in these isolates.
2. Materials and methods
2.1. Isolation and characterization of bacteria
During the year 2018, 286 faecal samples were taken from four
different healthy broiler in chicken farms located in Mateur (n = 110
samples), Zaghouan (n = 56 samples) (north of Tunisia), Grombalia
(n = 40 samples) and Soliman (n = 40 samples) (Nabeul governorate,
northeast coastal zone of Tunisia). On each farm, 60 to 75 single faecal
droppings were collected with sterile swaps and transported to the la-
boratory within 2–4 h. The mean average age of the broilers was
35 days. The farms were selected by considering their geographical
location and the size of the farms (at least 3000 chickens per farm). All
farms were under official control by the national veterinary services.
According to the veterinarians of those farms, Forkem (florfenicol),
Floxin (ofloxacin), and doxyvit (doxycycline) are usually used to treat
some infections specifically chronic respiratory diseases, colibacillosis,
or necrotic enteritidis. In addition, 47 raw chicken meat samples were
collected from various small stores (n = 26 samples; free outlets un-
controlled by hygienic services) and supermarkets (n = 21 samples;
controlled by national hygienic services) located in Tunis City during
various periods.
Approximately 5 g of each faecal sample and 25 g of each meat
sample were added in 75 mL and 225 mL of Buffered Peptone Water,
respectively, incubated at 37 °C for 24 h, and then streaked on Tryptone
Bile X-glucuronide (TBX) agar supplemented with 2 μg/mL of cefo-
taxime (CTX), followed by further incubation at 37 °C for 24 h. From
each sample, one colony with typical E. coli morphology (green-blue
colony) was selected and cultured overnight at 37 °C in nutritive agar.
Isolates were identified by MALDI-TOF MS (Bizzini and Greub, 2010).
2.2. Antibiotic susceptibility testing
Antibiotic susceptibility was performed by the disk diffusion
method according to the National Committee for Clinical Laboratory
Standards (CLSI, 2017) and the European Committee on Antimicrobial
Susceptibility Testing (EUCAST, http://mic.eucast.org/Eucast2/)
guidelines. Screening of ESBL production was performed by the Double
Disk Synergy Test (DDST) (CLSI, 2017). The determination of the
Minimum Inhibitory Concentration (MICs) for colistin was performed
using the broth micro-dilution method (BMD) (CLSI, 2017).
2.3. Detection of ESBL, tetracycline-, sulfonamide-, and chloramphenicol
encoding genes
DNA extraction was performed using the boiling method. The
blaTEM, blaSHV, blaCTX-M, and blaCMY genes were investigated using PCR
and sequencing as previously described (Jouini et al., 2007). Tetra-
cycline-, sulfonamide-, and chloramphenicol-resistant isolates were
screened by PCR for the tetA and tetB genes, sul1 and sul3 genes, and
cmlA gene, respectively (Sáenz et al., 2004). Colistin-resistant isolates
2
International Journal of Food Microbiology 318 (2020) 108478
were screened for the presence of mcr-1 to mcr-8 genes, as previously
described (Liu et al., 2016; Rebelo et al., 2018).
2.4. Occurrence of class 1 integrons and content of their variable regions
The int1 gene, as well as the 3′-conserved sequence of class 1 in-
tegrons (qacEΔ-sul1), were studied by PCR in all trimethoprim/sulfa-
methoxazole-resistance strains (Sáenz et al., 2004). Variable regions of
class1 integrons were determined using PCR and sequencing (Sunde,
2005).
2.5. Characterization of plasmids, molecular typing (PFGE, MLST) and
phylotyping of E. coli isolates
For mcr1-harbouring isolates, plasmids of IncP, IncHI2, IncI1, and
IncFIB incompatibility groups, known as the relevant carriers of ESBL-
and mcr1-encoding genes, were investigated by a PCR-based replicon
typing method as previously described (Carattoli et al., 2005).
The E. coli strains were classified into one of the four major phy-
logenetic groups (A, B1, B2, and D) using the quadruplex PCR method
proposed by Clermont et al. (2000). Pulsed field gel electrophoresis
(PFGE) patterns with the XbaI enzyme were analysed in experiments
following the method of Sáenz et al. (2004) for 59 selected isolates.
MLST was performed for selected isolates according to their antibiotic
resistance profiles (mainly the presence of mcr-1 gene), phylogenetic
group, gene content, geographical origin, and Inc-type content, as
previously reported (Liu et al., 2015). The DNA sequencing of the in-
ternal fragments of seven housekeeping genes (adk, fumC, gyrB, icd,
mdh, purA and recA) was performed and the allelic profile of each strain
was determined and the sequence type (ST) was assigned in accordance
with the E. coli MSLT website (http://enterobase.warwick.ac.uk/
species/ecoli/allele_st_search).
3. Results
3.1. Isolation and identification of E. coli
Out of 333 samples, 94 (28.2%) contained cefotaxime-resistant E.
coli isolates, derived from 64 of 286 faecal samples of chickens (22.4%)
(Table 1) and from 30 of 47 poultry meat samples (63.8%) (Table 2). It
is worth noting that all farms contained cefotaxime-resistant E. coli
isolates. One isolate per positive sample was selected for further char-
acterization.
3.2. Antimicrobial susceptibility testing
Ninety of 94 cefotaxime-resistant E. coli isolates (95.7%) were ESBL-
producers as shown by DDST. The remaining four isolates (4.3%), re-
covered from chicken faeces, showed a negative DDST, suggesting the
production of an aAmpC.
High resistance rates were observed for non-beta-lactam antibiotics,
being 69.1% for chloramphenicol, 86.1% for ciprofloxacin, 80.4% for
trimethoprim/sulfamethoxazole, 89% for sulfonamide, 97.8% for tet-
racycline, and 96.8% for nalidixic acid. Gentamicin resistance was
observed in only three isolates (3.1%); however, all isolates were sus-
ceptible to imipenem. Fifty-four isolates (57.4%) were resistant to co-
listin (34 from chicken faeces and 20 from raw meat) (MIC ranged from
4 to 32 μg/mL). Most isolates (98.9%) were multidrug resistant (re-
sistant to at least three different antibiotic families), mainly to quino-
lones, tetracycline, and trimethoprim/sulfamethoxazole.
3.3. Genes encoding cefotaxime- and colistin-resistance and other resistance
markers
The β-lactamase genes detected by PCR and sequencing in the 90
ESBL-positive E. coli isolates encoded the following enzymes: CTX-M-1
B. Hassen, et al. International Journal of Food Microbiology 318 (2020) 108478

Table 1
Characterization of β-lactamase-producing E. coli isolates recovered from chicken faeces samples (n = 64).
Number of Phylogroup/ST PFGE types Resistance profile for Beta-lactamases MICd for mcr-1 Plasmid (Inc) Other genes Area Lots
strains typeb (number non-β-lactam COL (mg/ detected
performed) antibioticsc L)

8 D/ST57 E1 (2) NAL, CHL, COL, TET, CTX-M-1 + 16 + P, FIB intI1, tetA, Zaghouan Lot 1
CIP, SXT, S TEM-1b sul1
3 D E2 (1) NAL, CHL, COL, TET, CTX-M-1 + 16 + P, FIB intI1, tetA, Zaghouan Lot 1
CIP, SXT, S TEM-1b sul1
7 D/ST69 E3 (2) NAL, COL, TET, CIP, CTX-M-1 + 16 + P, FIB intI1, tetA, Zaghouan Lot 1
SXT, S TEM-1b sul1
3 D/ST69 E4 (3) NAL, CHL, COL, TET, CTX-M-1 + 8 + P, FIB intI1, tetA, Zaghouan Lot 1
CIP, SXT, S, GEN TEM-1b sul1
1 D Not typeable (1) NAL, TET, CIP, SXT, S CTX-M-1 + ≤0.5 − ND intI1, tetA, Zaghouan Lot 1
TEM-1b sul1
1 B1 Not typeable (1) NAL, CHL, COL, TET, CTX-M-1 + 16 + P, FIB intI1, tetA, Zaghouan Lot 1
CIP, SXT, S TEM-1b sul1
3 B1/ST162 Not typeable (3) NAL, COL, TET, CIP, CTX-M-1 + 16 + P, FIB intI1, tetA, Zaghouan Lot 1
SXT, S TEM-1b sul1
1 A Not typeable (1) NAL, CHL, COL, TET, CTX-M-1 + 16 + P, FIB intI1, tetA, Zaghouan Lot 1
CIP, SXT, S TEM-1b sul1
1 A E5 (1) NAL, CHL, COL, TET, CTX-M-1 + 32 + P tetA Zaghouan Lot 1
CIP TEM-1b
3 A/ST2220 E6 (3) NAL, COL, TET, CIP, CTX-M-1 + 16 + P intI1, tetA, Zaghouan Lot 1
SXT, S TEM-1b sul1
3 A/ST10 E7 (3) NAL, CHL, COL, TET, CTX-M-1 16 + P tetA Zaghouan Lot 1
CIP
5 A E8 (1) NAL, CHL, TET, CIP, CTX-M-1 + ≤0.5 − ND intI1, tetA, Soliman, Lot 1
SXT, S TEM-1b sul1 Nabeul
1 B1/ST5686 Not typeable (1) NAL, CHL, COL, TET, CTX-M-1 + 16 + P, FIB intI1, tetA, Soliman, Lot 1
CIP, SXT, S TEM-1b sul1 Nabeul
8 D/ST997 E9 (2) NAL, CHL, TET, CIP, CTX-M-55 + ≤0.5 − ND tetA, sul3 Soliman, Lot 1
SXT, S TEM-1b Nabeul
10 D/ST997 E10 (2) NAL, CHL, TET, CIP, CTX-M-55 + ≤0.5 − ND tetA, sul3 Soliman, Lot 1
SXT, S TEM-1b Nabeul
1 B1 E11 (1) None CTX-M-1 1 − ND − Mateur Lot 1
1 B1/ST6488 Not typeable (1) NAL, CHL, CIP CTX-M-14 ≤0.5 − ND − Soliman, Lot 1
Nabeul
4a B2/ST6789 E12 (4) NAL, TET CMY-2 ≤0.5 − NDe tetA Mateur Lot 1

PFGE resolving XbaI-restriction fragments representative of the 18 profiles (E1–E12).

a
DDST negative.
b
MLST performed only in selected isolates.
c
NAL: nalidixic acid, CIP: ciprofloxacin, S: sulfonamide, SXT: trimethoprim-sulfamethoxazole, TET: tetracycline, GEN: gentamicin; CHL: chloramphenicol, COL:
colistin.
d
MIC: Minimum Inhibitory Concentration.
e
ND: not done.

with/without TEM-1 (65 isolates: 41 from faeces and 24 from raw
meat), CTX-M-55 with/without TEM-1 (18 from faecal samples, 3 from
raw meat), CTX-M-14 (one from faeces), and SHV-12 (three from
faeces). The four isolates showing a negative DDST test harboured the
blaCMY-2 gene.
A total of 54 E. coli isolates (34 of faecal samples and 20 of raw
meat) showed colistin MICs ranging from 4 to 32 μg/ml. Among them
52, all CTX-M-1-producers, carried the mcr-1 gene (being negative for
the other mcr genes tested). Nevertheless, two E. coli isolates, with co-
listin MIC of 4 and 8 μg/mL, were negative for all eight mcr alleles
tested.
Tetracycline resistance, detected in 92 isolates, was encoded by tetA
(n = 91) and tetB (n = 1) genes. The cmlA gene was detected in 12
among the 65 chloramphenicol-resistant isolates. Sulphonamide re-
sistance was encoded by sul1 (n = 57) and sul3 (n = 25) genes.
3.4. Characterization of integrons, variable regions and plasmids
The int1 gene was detected in 76 (80.9%) isolates, all of them ESBL-
producers and trimethoprim/sulfamethoxazole-resistant. The variable
regions of five int1-positive isolates from raw chicken meat samples
were sequenced and all contained a unique gene cassette array:
aadA1 + dfrA.
The IncI1, IncFIB, and IncP replicons were detected among the
mcr1-harbouring E. coli isolates, and different replicon combinations
were identified (Tables 1 and 2). The IncI1, IncF and IncFIB replicons
were co-harboured by seven blaCTX-M-1-positive E. coli isolated from raw
chicken meat. Twenty-six blaCTX-M-1-positive E. coli strains, from
chicken faeces, showed both IncFIB and IncP replicons.
3.5. Molecular typing (PFGE, MLST) and phylogenetic analysis
Out of the 94 E. coli isolates, 47 (50%), 34 (36.2%), 9 (9.6%), and 4
(4.3%) appeared to belong to the phylogroups D, A, B1, and B2, re-
spectively. The MLST analysis was applied for selected isolates ac-
cording to the PFGE cluster analysis, the presence of mcr-1 gene, phy-
logenetic group, and origin (farm, lot; supermarket). The following STs
were detected: 1) ST10-A, ST398-A, ST2220-A, ST5416-A, ST117-D,
ST57-D, ST69-D, ST162-B1 and ST5686-B1 (harbouring both blaCTX-M-1
and mcr-1 genes); 2) ST2973-A (in one SHV-12 and one CTX-M-55); 3)
ST997-D (in CTX-M-55 isolates); 4) ST6488-B1 (in one CTX-M-14 iso-
late); and 5) ST6789-B2 (in CMY-2 isolates) (Tables 1 and 2). It is in-
teresting to note that only ST57 lineage was detected among isolates
from raw chicken meat and chicken faeces samples.
Fifty-nine representative isolates were tested by PFGE and 50 of
them were typeable and were classified into 28 different pulsotypes
(assigned as E1–E28). Four, eight and 14 of the pulsotypes each in-
cluded three, two and one isolate, respectively. Two pulsotypes

3
B. Hassen, et al. International Journal of Food Microbiology 318 (2020) 108478

Table 2
Characterization of β-lactamase-producing E. coli isolated from chicken meat samples (n = 47).
Number of Phylogroup/ST PFGE types (num. Resistance profile for Beta-lactamases MICc of mcr-1 Plasmid Other genes detected Area
strains typea performed) non-beta-lactam COL (mg/ (Inc)d
antibioticsb L)

Free outlets
1 D/ST57 E13 (1) NAL, COL, CHL, TET, CIP, CTX-M-1 + 16 + P, I1 intI1, tetA, sul1 Tébourba1
SXT, S TEM-1b
3 A/ST398 E14 (3) NAL, COL, CHL, TET, CIP, CTX-M-1 + 16 + P, FIB, I1 intI1, tetA, cmlA, sul3 Zahrouni
SXT, S TEM-1b
1 A E15 (1) NAL, TET, CIP, SXT, S CTX-M-1 + ≤0.5 − ND intI1, qacEΔ1-sul1, Zahrouni
TEM-1b dfrA1, aadA1, tetA
1 A E16 (1) NAL, COL, CHL, TET, CIP CTX-M-1 8 + P, FIB tetA, cmlA Zahrouni
1 A E17 (1) NAL, COL, TET, CIP, SXT, CTX-M-1 16 + P, I1 intI1, tetA, sul3 Zahrouni 3
S
1 A ND NAL, CHL, TET, CIP, SXT, CTX-M-1 ≤0.5 − ND intI1, tetA, cmlA, sul3 Zahrouni 4
S
1 A E18 (1) NAL, CHL, TET, CIP, SXT, CTX-M-55 2 − ND intI1, tetA, sul1 Zahrouni
S

Controlled outlets (supermarket)

1 A/ST5416 E19 (1) NAL, COL, CHL, TET, CIP, CTX-M-1 + 8 + P, FIB tetA, cmlA, sul1 Zahrouni
S TEM-1b
1 A/ST5416 E19 (1) NAL, COL, CHL, TET, CIP, CTX-M-1 + 16 + P, FIB, I1 tetA, cmlA, sul1 Zahrouni
S TEM-1b
1 A E20 (1) NAL, COL, TET, CIP, SXT, CTX-M-1 + 16 + P, FIB intI1, qacEΔ1-sul1, Mannouba
S TEM-1b tetA, dfrA1, aadA1
1 A E21 (1) NAL, COL, CHL, TET, CIP, CTX-M-1 + 16 + P, FIB intI1, qacEΔ1-sul1, Wardia
SXT, S TEM-1b tetA, dfrA1, aadA1
1 A E21 (1) NAL, COL, TET, CIP, SXT, CTX-M-1 + 8 + P, FIB intI1, tetA, sul1 Tunis
S TEM-1b
1 A ND NAL, CHL, TET, SXT, S CTX-M-1 + ≤0.5 − ND intI1, tetA, sul3 Zouhour
TEM-1b
2 D/ST57 E22 (2) NAL, COL, CHL, TET, CIP, CTX-M-1 + 16 + P, FIB, I1 intI1, tetA, sul1 Wardia
SXT, S TEM-1b
1 D/ST57 E22 (1) NAL, COL, CHL, TET, CIP, CTX-M-1 + 16 + P, FIB, I1 intI1, tetA, sul1 Tunis
SXT, S TEM-1b
1 D/ST57 E22 (1) NAL, COL, CHL, TET, CIP, CTX-M-1 + 16 + P, FIB intI1, tetA, sul1 Tunis
SXT, S TEM-1b
1 D/ST117 E23 (1) NAL, COL, TET, S CTX-M-1 32 + P, FIB tetA, sul1 Zahrouni
1 D/ST117 E23 (1) NAL, COL, CHL, TET, S CTX-M-1 16 + P, FIB tetA, cmlA, sul1 Zahrouni
1 A E24 (1) NAL, COL, CHL, TET, CTX-M-1 16 + P, I1 intI1, qacEΔ1-sul1, Zouhour
SXT, S dfrA1, aadA1, tetA,
cmlA, sul3
1 A E25 (1) NAL, COL, TET CTX-M-1 4 − ND tetA Tunis
1 A E26 (1) NAL, TET, CIP CTX-M-1 1 − ND tetA Tunis
1 B1 ND NAL, TET, CIP, SXT, S CTX-M-55 1 − ND intI1, tetA, sul1 Tunis
1 A/ST2973 E27 (1) NAL, TET, CIP, SXT, S CTX-M-55 ≤0.5 − ND intI1, tetA, sul1 Tunis
1 A E28 (1) NAL, CHL, TET, CIP, SXT, SHV-12 2 − ND intI1, tetA, sul1 Mannouba
S
1 A Not typeable (1) NAL, CHL, TET, CIP, S SHV-12 2 − ND intI1, tetA, sul1 Mannouba
1 A/ST2973 E27 (1) NAL, COL, TET, CIP, SXT, SHV-12 8 − ND intI1, tetA, sul1 Tunis
S
1 B1 ND NAL, TET, CIP, SXT, S CTX-M-1 + ≤0.5 − ND intI1, qacEΔ1-sul1, Tunis
TEM-1b tetA, dfrA1, aadA1

PFGE resolving XbaI-restriction fragments representative of the 17 profiles (E13–E28).

a
MLST investigated only in selected isolates.
b
NAL: nalidixic acid, CIP: ciprofloxacin, S: sulfonamide, SXT: trimethoprim-sulfamethoxazole, TET: tetracycline, GEN: gentamicin; CHL: chloramphenicol, COL:
colistin.
c
MIC: Minimum Inhibitory Concentration.
d
ND: not done.

contained four isolates each. Nine isolates were not typeable by PFGE
despite several attempts (Tables 1, 2).
4. Discussion
ESBL- and aAmpC-producing E. coli isolates have been increasingly
reported worldwide from either human, animal, food products of an-
imal origin, environment (Dorado-García et al., 2017) or vegetables
(van Hoek et al., 2015). Humans could acquire ESBL/aAmpC-producing
E. coli via the food chain, and many reports have shown a genetic re-
lationship between such isolates of human and animal origins, espe-
cially from poultry and porcine isolates (Madec et al., 2015). In Tunisia,
many reports have investigated the occurrence of extended-spectrum
cephalosporin-resistant E. coli from animal origins (Alonso et al., 2017;
Sghaier et al., 2019), but many epidemiological traits should also be
clarified and need to be highlighted.
Throughout the year 2018, 286 faecal samples were collected from
different healthy broiler in chicken farms located in 3 different areas in
Tunisia (Mateur, Nabeul and Zaghouan), and 47 retail chicken meat
samples (26 from free outlets and 21 from controlled outlets [super-
markets]). A high frequency of cefotaxime-resistant E. coli were isolated
from poultry faecal samples (22.4%) and from raw poultry meat sam-
ples (63.8%); these findings appeared similar to those reported in
previous studies in Tunisia (Ben Sallem et al., 2012; Maamar et al.,

4
B. Hassen, et al.
2016), and worldwide (Botelho et al., 2015).
As described previously, most of our isolates were multidrug re-
sistant, including tetracycline (97.8%), ciprofloxacin (86.1%), tri-
methoprim/sulfamethoxazole (80.4%), sulfonamide (56.4%), and
chloramphenicol (69.1%). All these results are concordant with data
reported by other authors (Maamar et al., 2016; Sghaier et al., 2019).
Interestingly, half of ESBL-producing isolates were colistin resistant.
Moreover, 53.1% and 42.6% of isolates collected from poultry faeces
and raw poultry meat samples, respectively, were colistin-resistant
(mostly mcr-1-mediated). These percentages are higher than the ones
reported by other authors (Belaynehe et al., 2018; Maamar et al., 2018;
Moawad et al., 2018).
The blaCTX-M-1 gene, frequently associated to blaTEM-1b, is the
dominant ESBL type in E. coli of the intestinal microbiota of chickens
and chicken meat in our study and in others worldwide (Girlich et al.,
2007; Grami et al., 2013; Maamar et al., 2016; Sghaier et al., 2019).
Interestingly the blaCTX-M-55 gene, found in 21 ESBL-producing strains
(18 from faeces and 3 from raw chicken meat), has never been reported
in Tunisia in avian ESBL-producing E. coli (Grami et al., 2013; Maamar
et al., 2016; Sghaier et al., 2019). This gene remains scarce in Tunisia E.
coli isolates of human and animal origins. On the contrary, the blaCTX-M-
55 gene seems the most common and dominant gene in E. coli isolated in
China from domestic animals (Tong et al., 2015).
Furthermore, in our country, Jouini et al. (2007) and Maamar et al.
(2016) reported low rates of blaCTX-M-14-producing E. coli isolates from
chickens; similarly, this gene was found in only one isolate in our study.
The blaSHV-12 gene was detected in three ESBL-producing E. coli isolates
originating from chicken faeces, similar results were reported by Brinas
et al. (2003) from healthy chickens in Spain. Moreover, we detected
blaCMY-2 gene encoding AmpC β-lactamase in four cefotaxime-resistant
strains, this finding is comparable to those previously reported in Tu-
nisia by Ben Sallem et al. (2012) and Maamar et al. (2016).
Actually, the mcr-1 gene is highly reported worldwide in
Enterobacteriaceae commonly isolated from animals, foods of animal
origin and humans (Wang et al., 2017). Among 54 colistin-resistant
isolates, 52 harboured the mcr-1 gene in association with blaCTX-M-1 and
blaSHV-12 genes in 51 and one isolate, respectively. In a previous Tuni-
sian study (Grami et al., 2016) on healthy chicken, both genes (mcr-1+
blaCTX-M-1) were located on a single and large (250–280 Kb) IncHI2-type
plasmid. However, in another Tunisian report, the mcr-1 gene was de-
tected in CMY-2- harbouring E. coli isolated from poultry faeces, and
was carried by IncP plasmids with approximate molecular size of 242
Kb that did not harbour the blaCMY-2 gene (Maamar et al., 2018). It is of
interest to highlight that two isolates with MIC for colistin of 4–8 μg/mL
were negative for all 8 mcr genes tested; we cannot discard the plausible
presence in these strains of new mcr-variant(s), an aspect that should be
evaluated in the future. Classical chromosomal mutations might also
explain this colistin resistance.
The tetA (and occasionally tetB) was the predominant gene among
tetracycline-resistant E. coli strains in our study, as well as in various
ecological niches (Soufi et al., 2011).
The high frequency of detection of class 1 integrons among cefo-
taxime-resistant isolates (80.9%), in most cases associated to the sul1-
gene is noteworthy, although integrons linked to the sul3 gene (non-
classic-class1 integrons) were also detected, were referred in other
studies (Soufi et al., 2011; Jiang et al., 2017). Most ESBL-producing E.
coli in our study belonged to phylogroup D (50%), and A (36.2%), and
lower rates were detected in relation to group B1 (9.6%). E. coli of
phylogroup B2 were only identified in the four CMY-2-positive isolates
of chicken faeces (4.3%), and in none of ESBL-positive isolates; never-
theless, this phylogroup is frequently detected among clinical ESBL-
positive isolates (Ben Sallem et al., 2012).
The genetic lineages of representative isolates according to their bla
genes, origins, phenotypes of colistin resistance and phylogenetic
groups were determined. Different genetic lineages carried both the
blaCTX-M-1 and mcr-1 genes were observed, being A-ST398, A-ST10, B1-
5
International Journal of Food Microbiology 318 (2020) 108478
ST162, D-ST57 and D-ST117. Isolates of those lineages carrying the
blaCTX-M-1 gene have been described in E. coli from different countries,
highlighting the wide spread of these international clones (Ben Sallem
et al., 2014; Zhang et al., 2016). Moreover, the CMY-2-producing
strains of phylogroup B2 corresponded to ST6798.
The presence of selected replicon types was studied in mcr-1/blaCTX-
M-1 positive E. coli isolates of this study and all of them carried the IncP
replicon, associated in 35 isolates to IncFIB, in 7 isolates to
IncFIB + IncI1 and in 3 isolates to IncI1. Recently, an IncP plasmid was
reported to be associated with the colistin resistance gene mcr-1 and its
variant mcr-1.6 (Rozwandowicz et al., 2018). A previous study per-
formed in Tunisia on E. coli isolates of chicken origin, detected the mcr-
1 gene located in an IncP plasmid of about 250 Kb, separated of the
blaCMY-2 gene also present in the strain (Maamar et al., 2018); although
other authors detected the mcr-1 gene in CTX-M-1-positive chicken E.
coli isolates in Tunisia into an IncHI2 plasmid (Grami et al., 2016).
The CTX-M-1, CMY-2 and SHV-12 types are frequently associated
with IncI1 plasmids (Accogli et al., 2013), and mainly co-harboured
genes encoding sulfonamide and trimethoprim resistance (Ben Sallem
et al., 2014; Hopkins et al., 2006).
5. Conclusion
In conclusion, we report in this study the widespread diffusion of
CTX-M-1-producing E. coli in poultry and in poultry meat as well as mcr-
1 gene. MLST showed the presence of different lineages carrying ESBL
and mcr-1 genes, mainly of some international clones. The occurrence
of plasmids belonging to specific Inc-types (IncI1-IncFIB-IncP) high-
lighted the dynamic genomes of avian ESBL-producing E. coli in
Tunisia. We also reported in this study for the first time the identifi-
cation of CTX-M-55-producing E. coli from healthy chicken.
Declaration of competing interest
The authors declare that there are no conflicts of interest.
Acknowledgment
The authors would like to express great appreciation to all of the
Tunisian and Spanish teams that effectively participated in the pre-
paration of this scientific research. The authors also thank the farmers
for their important collaboration in sampling.
Financial support
The work performed in the University of La Rioja was financed by
project of Agencia Estatal de Investigación (AEI) of Spain (SAF2016-
76571) and FEDER of EU. B. Hassen has a fellowship from the Tunisian
Ministry of Higher Education and Scientific Research (Tunisia). L. Ruiz-
Ripa has a predoctoral fellowship of the University of La Rioja (Spain).
O. M. Mama has a predoctoral fellowship of Mujeres por África-
Universidad de La Rioja.
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