Veterinary Microbiology 270 (2022) 109453

Contents lists available at ScienceDirect

Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Clonal and plasmid-mediated flow of ESBL/AmpC genes in Escherichia coli

in a commercial laying hen farm
Irene Aldea a, Alicia Gibello b, Marta Hernández c, Pimlapas Leekitcharoenphon d,
Valeria Bortolaia e, Miguel A. Moreno a, b, *
a
Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense de Madrid, Madrid, Spain
b
Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain
c
Laboratorio de Biología Molecular y Microbiología, Instituto Tecnológico Agrario de Castilla y León (ITACYL), Valladolid, Spain
d
Research Group for Genomic Epidemiology, National Food Institute, Technical University of Denmark, Lyngby, Denmark
e
Department of Bacteria, Parasites and Fungi, Statens Serum Institut, Copenhaguen, Denmark

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

Keywords: Resistance to third- and fourth-generation cephalosporins in Escherichia coli is mainly due to extended-spectrum
CTX-M-1 beta-lactamases (ESBL) and AmpC cephalosporinases, which have been increasingly reported, mainly in isolates
SHV-12 from humans and poultry.
CMY-2
The aim of this study was to address the flow of antimicrobial resistance determinants in the full laying hen
Egg production
Dynamics
production cycle (four batches followed from day-old chicks to 83/84-week-old layers), using cephalosporin-
Cephalosporin resistance resistant E. coli as a model and their characterization using whole genome sequencing (WGS).
Fifteen out of 22 samples analysed yielded growth on MacConkey agar with cefotaxime (1 mg/L). Of these,
141 isolates were identified as E. coli and 47 were characterized by WGS.
Genes detected were three ESBL (blaCTX-M-1 (n = 19); blaCTX-M-14 (n = 1); and blaSHV-12 (n = 9)) and one AmpC
(blaCMY-2 (n = 13)). Some isolates only harboured blaTEM-1B (n = 2) or blaTEM-1D (n = 1).
IncI1 plasmids were the main platform for ESBL/AmpC genes. In addition, five clones were identified har­
bouring blaCTX-M-1 (two), blaSHV-12 (one) and blaCMY-2 (two), drawing a clone-plasmid mixed flow model.
Gene blaCTX-M-1 was found in the chromosomal DNA of clone 1 over 14 months, and in IncI1/ST3 plasmids over
six months; over six months blaSHV-12 was found harboured by clone 3 (IncI1/ST26 plasmids), and 15 months
later in a non-replicon detected plasmid. Finally, blaCMY-2 spread for at least 16 months, mainly by IncK2
(including clone 4) and IncI1/ST12 (clone 5) plasmids.
Proper use of antimicrobials should be combined with other farm management strategies for the effective
control of cephalosporin-resistant E. coli isolates in commercial layer farms.

1. Introduction Surveillance Programme, we reported the first detection of two ESBL

(blaSHV-12 and blaCTX-M-14) and one AmpC (blaCMY-2) genes in E. coli from
Beta-lactams are a broad family of antimicrobials that include the broilers. Since then, ESBL/AmpC-producing E. coli have been increas­
cephalosporins. Escherichia coli resistance to third- and fourth- ingly detected, mainly from humans and healthy animals, especially
generation cephalosporins is mainly due to extended-spectrum beta- poultry. Consequently, poultry (mainly broilers and to a lesser extent
lactamases (ESBL) and AmpC cephalosporinases, which are generally laying hens) became an important reservoir for these resistance traits
plasmid-encoded. Resistance to cephalosporins in E. coli can also be (Saliu et al., 2017), posing a public health problem as a potential source
mediated by chromosomal point mutations in the ampC promoter of AMR bacteria in humans after consuming meat or eggs. Although
causing its overexpression. studies of ESBL/AmpC E. coli in eggs are rare, both positive (presence of
Nearly 20 years ago, as part of the Spanish Antimicrobial Resistance E. coli harbouring blaCTX-M-2 in eggs (Grande Burgos et al., 2016) and

* Correspondence to: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro, s/n, Madrid 28040,
Spain.
E-mail address: [email protected] (M.A. Moreno).

https://doi.org/10.1016/j.vetmic.2022.109453
Received 28 July 2021; Received in revised form 9 March 2022; Accepted 9 May 2022
Available online 16 May 2022
0378-1135/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
I. Aldea et al. Veterinary Microbiology 270 (2022) 109453

E. coli with ESBL phenotype in raw-egg surfaces (Rasheed et al., 2014) batches 1 (at week 24) and 2 (at week 23). Only samplings one to four,
and negative (no detection Stewardson et al., 2014) results have been and seven or eight (only the last grew in MacConkey agar with cefo­
reported. taxime [CTX]) have been included in this study.
The types of ESBL-producing Enterobacteriaceae and their occur­
rence in poultry has been reviewed (Saliu et al., 2017), and many studies 3. Bacterial isolation, identification and characterization
have reported the detection of cephalosporin-resistant E. coli in healthy
poultry, including layers, and some in clinical isolates (Niero et al., Peptone water-diluted samples (10 g in 90 mL) were maintained at
2018). Most of these studies are cross-sectional or surveillance studies room temperature for one hour before being serially diluted tenfold with
focused on cephalosporin-resistant E. coli detection in healthy broilers peptone water. Then, 0.1 mL of three dilutions (usually the first, second
(revised by Saliu et al., 2017), longitudinal studies following and third dilutions) were plated on MacConkey agar plates containing
cephalosporin-resistant E. coli transmission across the broiler production CTX at 1 mg/L and incubated at 37 ºC for 20–24 h. Ten lactose positive
pyramid (Apostolakos et al., 2019; Dame-Korevaar et al., 2017; Mo isolates of different morphology, if any, were taken from different plates
et al., 2014; Zurfluh et al., 2014), or longitudinal studies at the farm and dilutions, and PCR bacterial identification was performed as
level on the dynamics of CTX-phenotypes (Baron et al., 2018). Although described previously (Moreno et al., 2019).
there are also some studies focused on or including laying hens (Baez The E. coli isolates were tested for susceptibility to antimicrobials by
et al., 2021; Blaak et al., 2015; Ceccarelli et al., 2019; Seo and Lee, 2019; broth microdilution using EUVSEC commercial plates (Sensititre@,
among others), we found no longitudinal studies of the dynamics of Thermofisher) (Moreno et al., 2019). EUCAST epidemiological cut-off
different AMR-phenotypes at the farm level in layers. values were used to interpret the minimum inhibitory concentration
The aim of this study was to address the flow of AMR determinants in (MIC) values. Only no wild-type isolates for CTX (MIC >= 0.25 mg/L) or
the full laying hen production cycle using cephalosporin-resistant E. coli ceftazidime (MIC <= 0.50 mg/L) were selected for analysis.
as a model. Having in mind that the use of antimicrobials throughout
this commercial cycle is usually scarce, other additional factors should 4. Whole genome sequencing (WGS) and bioinformatic analysis
be involved if a flow could be detected.
A subset of 46 isolates was sent to the Laboratorio Tecnológico
2. Methods Agrario de Castilla y León (ITACYL) for WGS analysis, which was per­
formed as described elsewhere (Moreno et al., 2019) and summarized in
Details about setting, sampling and sample preparation have been Supplementary file 1. Forty-five isolates were chosen according to the
published elsewhere (Moreno et al., 2019). Briefly, four batches (B1, B2, number of different AMR phenotypic profiles observed in each sampling
B3 and B4) of hens raised in a commercial laying hen farm in Spain were (at least half of the AMR phenotypic profiles of each sampling were
followed from day-old chicks to 83–84-week-old hens, performing eight represented in the sequenced isolates). The remaining was a
litter samplings (from S1 to S8) from March 2016 to October 2018 CTX-resistant isolate (L3M1-CIP10) recovered in MacConkey agar plates
(Table 1). The farm had two facilities separated by about five kilometres: supplemented with ciprofloxacin (0.125 mg/L). In addition, a previ­
one grouped the two growing sites (GS-A and GS-B) and the other the ously sequenced CTX-resistant isolate (L1M3–09), recovered from the
four laying houses (LH-C, LH-D, LH-E and LH-F). No antibiotics were farm using MacConkey agar plates with no antibiotic (Moreno et al.,
used during this longitudinal study, but two five—day colistin treat­ 2019), was added to the study.
ments were recorded, as previously reported (Moreno et al., 2019), in Multilocus sequence typing (ST) profiles were predicted using MLST

Table 1
Summary of data regarding samplings and E. coli isolation (MacConkey with cefotaxime 1 mg/L), AMR phenotyping and sequencing, from a commercial laying hen
farm.
Batch Sampling Animal type Sampling Site Sampling Nº of isolates and (antibiograms Number of AMR phenotypic profiles and (sequenced
week date performed) isolates)

B1 S1 Day-old 0 GS-A 18/03/2016 No growth

chicks
B1 S2 Pullets 2 GS-A 05/04/2016 10 (10) 1 (1)
B1 S3 Pullets 14 GS-A 24/06/2016 10 (6) 2 (2)
B1 S4 Laying hens 24 LH-C 08/09/2016 10 (10) 1 (2)
B1 S8 Laying hens 83 LH-C 30/10/2017 6 (6) 3 (2)
B2 S1 Day-old 0 GS-B 27/05/2016 No growth
chicks
B2 S2 Pullets 2 GS-B 13/06/2016 7 (6) 1 (2)
B2 S3 Pullets 15 GS-B 14/09/2016 10 (10) 3 (5)
B2 S4 Laying hens 24 LH-D 15/11/2016 10 (10) 6 (5)
B2 S7 Laying hens 68 LH-D 25/09/2017 9 (9) 3 (2)
B2 S8 Laying hens 83 LH-D 09/01/2018 No growth
B3 S1 Day-old 0 GS-B 11/10/2016 7 (7) 3 (3)
chicks
B3 S2 Pullets 2 GS-B 25/10/2016 10 (10) 5 (5)
B3 S3 Pullets 14 GS-B 17/01/2017 10 (10) 3 (4)
B3 S4 Laying hens 23 LH-E 22/03/2017 3 (3) 2 (2)
B3 S8 Laying hens 85 LH-E 06/06/2018 9 (9) 3 (2)
B4 S1 Day-old 0 GS-B 13/03/2017 No growth
chicks
B4 S2 Pullets 2 GS-B 27/03/2017 No growth
B4 S3 Pullets 13 GS-B 15/06/2017 10 (10) –
B4 S4 Laying hens 23 LH-F 22/08/2017 10 (10) 5 (7)
B4 S7 Laying hens 67 LHS- 02/07/2018 10 (10) 2 (1)
F
B4 S8 Laying hens 82 LH-F 15/10/2018 No growth

GS: Growing site. LH: Laying house

2
I. Aldea et al. Veterinary Microbiology 270 (2022) 109453

v2.0. Non previously detected ST types were submitted to EnteroBase 6. Antimicrobial susceptibility
(https://enterobase.warwick.ac.uk/). AMR genes and chromosomal
point mutations were studied using ResFinder software v.4.1 that con­ Antimicrobial susceptibility tests were performed on 136 isolates.
tains PointFinder software, selecting as parameters 90% of threshold for Fourteen isolates had CTX MIC values below 0.5 mg/L and were
%ID and 60% of minimum length for both applications. The search for excluded from the study, except for one that had a ceftazidime MIC
integrases was performed with blastx. Plasmidfinder v2.1 and pMLST value of 1 mg/L. The remaining 122 isolates had CTX MIC values of over
v2.0 were used to detect plasmid origin of replication and to predict 2 mg/L and ampicillin MIC values of over 64 mg/L. In 89 of these 122
plasmid multilocus sequence type, respectively. isolates, ceftazidime MIC values were equal to or higher than 1 mg/L,
The phylogenetic analysis was performed with CSI Phylogeny v.1.4 while in three of them MIC values below 1 mg/L. In all the 123 isolates,
(Kaas et al., 2014), and the resulting phylogenetic trees were uploaded meropenem MIC values were below 0.12 mg/L. MIC values observed for
to iTOL v4 (Letunic and Bork, 2019) for annotation. Isolates differing by colistin and tigecycline were below 2 mg/L and 16 mg/L, respectively.
40 or fewer single nucleotide polymorphisms (SNPs) were considered In addition, 103 isolates presented a multidrug resistance profile,
clones. being resistant to at least three classes of antimicrobials.
The CONTIGuator pipeline (Galardini et al., 2011) was used to map Nineteen phenotypic AMR profiles were observed among these 123
contigs against reference sequences of the putative plasmids and inte­ isolates. The number of different AMR profiles per sample ranged from
grons detected. These reference sequences, when available, were chosen one to six, with the lowest variability found in batch 1 (Table 2). The
from poultry E. coli. Then, blastn was used for an additional comparison AMR profile most frequently observed included resistance to quinolones
of the sequences obtained from the CONTIGuator using the previous (ciprofloxacin and nalidixic acid) and sulphonamides, in addition to
reference sequences to check for identity and coverage. resistance to beta-lactams (ampicillin, cefotaxime, and ceftazidime), and
was detected in all batches and samplings, except sampling 8. At least 27
5. Results AMR phenotypic profiles were detected in two or more isolates from the
same sample, and 10 isolates with the same AMR phenotypic profile
5.1. Detection of cephalosporin-resistant E. coli were detected in batch 1 (samplings two and four) (Table 2).

Only samples from one of the four batches (batch 3) showed bacterial 7. Molecular characterization of cephalosporin-resistant E. coli
growth on CTX selective medium in samples from day-old chicks.
Nevertheless, samples from all but one batch (batch 4) had growth on Among the 47 sequenced isolates we detected fifteen previously
this medium in the second (two week pullets) and third samplings (14- described STs, being ST155 (n = 9), ST93 (n = 8) and ST4243 (n = 6) the
week pullets). most frequent. In addition, two isolates were assigned to novel STs
All batches showed growth in CTX selective medium in samples from named ST13021 (close to ST155) and ST 13030 (close to ST170),
23 to 24-week laying hens, but only batches 1 and 3 showed growth in whereas one isolate cannot be fully assigned and was recorded as close
the last sampling (82–85-week hens) (Table 1). to ST1818.
Overall, 15 out of 22 samples analysed yielded growth on CTX se­ The IncI1 replicon type was the most detected (IncI1–3, n = 10;
lective medium. From these, a total of 141 isolates were recovered and IncI1–26, n = 7; IncI1–12, n = 3), followed by IncX1 (n = 13), IncK2 (n
identified as E. coli. = 10), IncA/C2 (n = 5) and IncFII (n = 3). Seven isolates harboured
IncI1–26 + IncX1 and five IncX1 + IncA/C2.

Table 2
Distribution by batch, sampling and antimicrobial resistance (AMR) profile of 124 E. coli isolated in medium containing cefotaxime (CTX) at 1 mg/L, from a com­
mercial laying hen farm.
AMR profile Batch B1 B2 B3 B4

Sampling S1 S2 S3 S4 S8 S1 S2 S3 S4 S7 S1 S2 S3 S4 S8 S1 S2 S3 S4 S7

AMP-CTX/TAZ-CIP/NAL-SMX-TET- 4 3 1 4 9 3
TMP
AMP-CTX/TAZ-CIP/NAL-SMX 10 2 4 3 1 1
AMP-CTX/TAZ-SMX-TET 10 4 4 1
AMP-CTX/TAZ 6 1 1 1
AMP-CTX/TAZ-CIP-SMX-TET-CHL 3 3 2
AMP-CTX/TAZ-CIP/NAL-SMX-TET 2 1 1
AMP-CTX/TAZ-SMX-TET-TMP 5 1
AMP-CTX/TAZ-SMX-CHL 2 3
AMP-CTX-SMX-TET 1 2
AMP-CTX/TAZ-SMX-TET-AZI 1 1
AMP-CTX/TAZ-SMX 6
AMP-CTX/TAZ-CIP/NAL 3
AMP-TAZ-TET 3
AMP-CTX-SMX-TET 1
AMP-CTX/TAZ-SMX-TET-TMP 1
AMP-CTX/TAZ-CIP/NAL-SMX-TET- 2
CHL
AMP-CTX/TAZ-CIP/NAL-SMX-TMP 2
AMP-CTX/TAZ-CIP/NAL-SMX-TET- 2
TMP-GEN-AZI
AMP-CTX/TAZ-CIP/NAL-SMX-TET- 1
TMP
AMP-CTX-CHL-CIP-SMX-TET-GEN 1
AMP-CTX-CIP/NAL-SMX-TET-TMP 1

AMP: ampicillin; TET: tetracycline; SMX: sulfonamides; CIP: ciprofloxacin; NAL: nalidixic acid; TMP: trimethoprim; CHL: chloramphenicol; GEN: gentamicin; CTX:
cefotaxime; TAZ: ceftazidime; AZI: azithromycin.

3
I. Aldea et al. Veterinary Microbiology 270 (2022) 109453

In the 47 isolates sequenced, cephalosporin resistance was mainly integron carrying aadA5 (streptomycin/spectinomycin resistance) and
associated with ESBL/pAmpC-encoding genes. The genes detected in dfrA1 (trimethoprim resistance) genes.
this study were three ESBL (blaCTX-M-1 [n = 19]; blaCTX-M-14 [n = 1]; and
blaSHV-12 [n = 9]) and one pAmpC (blaCMY-2 [n = 13)]. In addition, three 8. Dynamics of blaCTX-M-1 in the farm
types of mutations (− 18 and − 1, always found together, [n = 14], and
+24 [n = 8]) were found in the ampC gene and/or its promoter (n = 22). Gene blaCTX-M-1 was the most frequently detected ESBL gene (n = 19
In all cases, these mutations were found in isolates harbouring ESBL/ isolates) in the farm, among which we identified two clones (Fig. 1).
pAmpC genes. Three isolates only harboured blaTEM-1B (n = 2) or blaTEM- Clone 1 comprised eight ST93 isolates harbouring the gene in the
1D (n = 1), although these bla genes were also detected in 15 of the chromosome, while clone 2 (three ST117 isolates) harboured the gene in
previously mentioned isolates: blaTEM-1B in combination with blaCTX-M-14 IncI1/ST3 plasmids. Gene blaCTX-M-1 was also located in IncI1/ST3
(n = 1), blaSHV-12 (n = 7) and blaCMY-2 (n = 2), and blaTEM-1D in com­ plasmids in eight additional isolates with other ST profiles (Table 3).
bination with blaCTX-M-1 (n = 5). Into the remaining isolate we did not All the blaCTX-M-1 (in plasmids IncI1/ST3 or chromosomes) contain­
detect either bla/AmpC genes or AmpC mutations. ing isolates harboured an ISEcp1 element located in the upstream region
The gene blaCTX-M-14 was identified in the chromosomal DNA of one of the blaCTX-M-1 gene. CONTIGuator assembled contigs of 11 isolates
ST2599 isolate (batch 2/S4, 67-week laying hens/laying house D). The containing blaCTX-M-1 plus ISEcp1 (size between 110,613 and
isolate also carried blaTEM-1B and the sulphonamide- and tetracycline- 121,618 bp) in IncI1/ST3 plasmids, were compared with the blaCTX-M-1
resistance genes tetA and sul2, respectively, as well as a class 1 plus ISEcp1 sequence of a published IncI1/ST3 plasmid (Accession no.

Fig. 1. Phylogenetic tree of the 47 Escherichia coli isolates from a commercial layer hen farm, E. coli K12 was used as reference genome, First column: batch and
sampling; second colum: ST profile; third column: ESBL/pAmpC gene detected; fourth column: plasmid replicon; subsequent columns: presence (green)/absence
(white) of point mutations in the AmpC promotor and antibiotic resistance genes, Isolates differing by 40 or fewer single nucleotide polymorphisms (SNPs) were
considered clones and are represented with a red dot. Clone 1 corresponds to blaCTX-M-1/ST93 isolates; clone 2 corresponds to blaCTX-M-1/ST117 isolates; clone 3
corresponds to blaSHV-12/ST155 isolates; clone 4 corresponds to blaCMY-2/ST4243 isolates; and clone 5 corresponds to blaCMY-2/ST4038 isolates.

4
I. Aldea et al. Veterinary Microbiology 270 (2022) 109453

Fig. 1. (continued).

KM377240) from broiler E. coli from Switzerland (Zurfluh et al., 2014), in IncI1/ST3 plasmids over six months (two batches) harboured by iso­
and 98%− 100% were identical (93%− 96% coverage). lates with three different MLST profiles, including clone 2.
All the 11 IncI1/ST3 plasmids also harboured sul2 and tetA. In
addition, one of the isolates (ST1716) harbouring this plasmid also had a 9. Dynamics of gene blaSHV-12 in the farm
class 1 integron, located in a non-typeable plasmid, containing dfrA1
and aadA1 (streptomycin/spectinomycin resistance). In the farm, the gene blaSHV-12 was identified in nine isolates
All the isolates of clone 1 had a class 1 integron, carrying resistance belonging to two different ST types (ST115 and 6616) and harboured by
genes dfrA1, aadA1 and sul1 (sulphonamides resistance). This integron two different plasmids (Table 3). Seven ST155 isolates harboured the
was detected in two different plasmid types (IncA/C2 and IncFII) gene on IncI1/ST26 plasmids and were grouped as clone 3 (Fig. 1),
(Table 3). Six of these isolates (from batches 1, 2 and 4) also had an whereas the two remaining isolates belonged to ST6616 and harboured
IncX1 plasmid harbouring tetA and blaTEM-1D. the gene on non-typeable plasmids. blaSHV-12 in IncI1/ST26 plasmids
The chronology of the platforms where blaCTX-M-1 was detected in the was associated with an IS26 insertion sequence and the CONTIGuator
farm can be followed in Table 3. Clone 1 was first identified in the farm assembled contigs (size between 115,209 and 130,170 bp) of seven
in batch 1/S3 (14-week pullets/growing site A). Three months later, it isolates showed 99% similarity (96%− 98% coverage) with the same
was identified in batch 2/S3 (14-week pullets/growing site B), and two structure described in plasmid pCAZ590 from broiler E. coli (Alonso
months later in the 24-week hens (S4) of this batch that were moved to et al., 2017) (Accession no. LT669764).
laying house D. The IncI1/ST26 plasmids also harboured tetA and a class 1 integron
This clone was again identified two months later in isolates of batch carrying aadA1 and aadA2, the sulfonamide resistance gene sul3, and the
3/S3 (14-week pullets/growing site B). Finally, it was identified in two chloramphenicol resistance gene cmlA1.
isolates of batch 4/S4 (23-week hens/laying house F). The sequence of the class 1 integron of six isolates was compared
The IncI1/ST3 plasmid containing the gene blaCTX-M-1 was firstly with a published isolate (Accession no. HQ875017) identified in IncI1
detected in the farm in ST5853 isolates from batch 1/S4 (24-week hens/ plasmids in ESBL E. coli (Curiao et al., 2011), showing 100% coverage
laying house C) (Table 3). One month later, it was detected in isolates of and identity.
three different ST profiles (ST155, 602 and 117/clone 2) of batch 3/S1 All the isolates belonging to clone 3 had an additional IncX1 plasmid,
(day-old chicks/growing site B), and again in the subsequent sampling harbouring blaTEM-1B and the fluoroquinolone resistance gene qnrS1, and
(S2) of this batch (two week pullets/growing site B) in three isolates the two ST6616 isolates also had tetA and a chromosomal class 1 inte­
(ST10, 1716 and 117/clone 2) (Table 3). gron harbouring dfrA1, aadA1 and sul1.
Five months later, the IncI1/ST3 plasmid containing the gene blaCTX- The chronology of blaSHV-12 in the farm showed that clone 3 was first
M-1 was detected in the same batch 3/S4 in a ST117/clone 2 isolate (23- identified in batch 2/S3 (15-week pullets/growing site B), then at week
week laying hens/laying house E). 24 (S4) in the same batch moved to a laying house (D), and again four
Finally, seven months later, it was identified in an isolate with an months later in batch 3/S4 (23-week laying hens/laying house E).
ST close to 155 in batch 1/S8 (83-week hens/laying house C). Therefore, the blaSHV-12 gene was found in batches 2 and 3 over six
In summary, blaCTX-M-1 gene has been found in chromosomal DNA in months harboured by clone 3 in the same type of plasmids type and 15
clonal ST93 isolates over 14 months in the farm in the four batches, and months later in a no-typeable plasmid in ST 6616 isolates from 83-week

5
I. Aldea et al. Veterinary Microbiology 270 (2022) 109453

Table 3 Table 3 (continued )

Distribution by origin and features of the 42 E. coli isolates containing ESBL/ Batch/ ST ESBL/ AmpC Plasmid Plasmid
pAmpC genes from a commercial layer hen farm in Spain. Sampling/ AmpC mutations replicon replicons
Batch/ ST ESBL/ AmpC Plasmid Plasmid site gene (nn) harbouring containing
Sampling/ AmpC mutations replicon replicons Isolation (n) ESBL/AmpC other AMR
site gene (nn) harbouring containing date (other AMR genes and/
Isolation (n) ESBL/AmpC other AMR genes and/or or integrons
date (other AMR genes and/ integrons) (nn)
genes and/or or integrons B3/S4/E 155 aadA1-aadA2- IncX1
integrons) (nn) 22/03/ (Clone sul3-cmlA1)] (qnrS1-
B1/S2/A 937 blaCMY- -18, − 1 IncK2 2017 3) blaTEM-1B)
05/04/ 2
117 blaCTX- +24 IncI/ST3 (sul2-
2016 (Clone M-1 tetA)
B2/S2/B 1158 blaCMY- IncK2 2) *
16/06/ 2
B4/S4/F 93 blaCTX- Chromosomal IncA/C2
2016 22/08/ (Clone M-1 (3) (int1-dfrA1-
B1/S3/A 4243 blaCMY- +24 (1) IncK2 2017 1) (3) aadA1-sul1)
24/06/ (Clone 2 (2) + IncX1
2016 4) (blaTEM-1D
93 blaCTX- Chromosomal IncA/C2 + tetA) (2)
(clone M-1 (int1-dfrA1- IncFII (IntI1-
1) aadA1-sul1) dfrA1-
+ IncX1 aadA1-sul2)
(blaTEM-1D (1)
+ tetA) 4243 blaCMY- +24 IncK2
B1/S4/C 5853 blaCTX- -18, − 1 IncI/ST3 (sul2- (Clone 2

08/09/ M-1 (2) tetA) 4)

2016 4038 blaCMY- -18, − 1 IncI1/ST12
B2/S3/B 155 blaSHV- -18, − 1 (2) IncI1/ST26 IncX1 (Clone 2 (2)

14/09/ (Clone 12 (3) [tetA + (IntI1- (qnrS1- 5)

2016 3)* * aadA1-aadA2- blaTEM-1B) B2/S7/F 2599 blaCTX- -18, − 1 Chromosomal
sul3-cmlA1)] 25/09/ M-14

4243 blaCMY- +24 IncK2 2017

(Clone 2
B1/S8/C 13021 blaCTX- -18, − 1 IncI/ST3 (sul2-
4) 30/10/ ** M-1 (2) tetA)
93 blaCTX- Chromosomal IncA/C2 2017
(Clone M-1 (int1-dfrA1- B3/S8/C 6616 blaSHV- No-replicon
1) aadA1-sul1) 06/06/ 12 (2) detected
+ 2018 plasmid
IncX1
n = number of isolates if higher than 1; nn = number of isolates if different than
(blaTEM-1D
n; * / **= 1 or 2 isolates with plasmid replicon and ESBL/pAmpC gene into the
+ tetA)
B3/S1/B 155 * blaCMY- IncK2 same contig
11/10/ 2
2016 155 * blaCTX- -18, − 1 (2) IncI/ST3 (sul2- layers (batch 3/S8/ laying house E).
117 M-1 (3) tetA)
(Clone
2) * 10. Dynamics of blaCMY-2 in the farm
602 -18, − 1
B3/S2/B 48 ** blaCMY- IncK2 The AmpC beta-lactamase gene blaCMY-2 was the second most widely
25/10/ 2 (2) identified in the farm, and was mainly located in IncK2 plasmids har­
2016 10 blaCTX- IncI/ST3 (sul2-
117
boured by five isolates of ST4243 (clone 4), two ST48 isolates and one
M-1 (3) +24 tetA)
(Clone No-replicon isolate of ST1158, ST155 and ST973, respectively (Table 3). It was also
2) * detected was detected in IncI1/ST12 plasmids harboured by three ST4038 iso­
1716 plasmid lates (clone 5).
B2/S4/D 155 blaSHV- -18, − 1 (2) IncI1/ST26 IncX1
Gene blaCMY-2 in IncK2 plasmids was located downstream to an
15/11/ (Clone 12 (3) [tetA + (IntI1- (qnrS1-
2016 3) aadA1-aadA2- blaTEM-1B) ISECp1 insertion sequence and upstream to genes blc and sugE, encoding
sul3-cmlA1)] a lipoprotein and a guanidinium exporter, respectively. The
93 blaCTX- Chromosomal IncA/C2 CONTIGuator-assembled sequences containing this arrangement in
(Clone M-1 (int1-dfrA1- IncK2 plasmids (10 isolates) were compared with those of the plasmid
1) aadA1-sul1)
+ IncX1
pDV45 from Italian broiler E. coli (Apostolakos et al., 2020a) (Accession
(blaTEM-1D no. KR905384.1), obtaining 99% similarity (90%− 100% coverage) with
+ tetA) the ISEcp1-blaCMY-2-blc-sugE structure.
4038 blaCMY- -18, − 1 IncI1/ST12 IncI1/ST12 plasmids harbouring blaCMY-2 had an ISECp1 element
(clone 2
upstream and the CONTIGuator-assembled sequences (three isolates)
5) *
B3/S3/B 93 blaCTX- Chromosomal IncFII (IntI1- were compared with a similar structure described by Roer et al. (2019)
17/01/ (Clone M-1 (2) dfrA1- (Accession no. MH472638), obtaining 93% similarity (100% coverage).
2017 1) aadA1-sul2) The three ST4038 isolates (clone 5) also harboured non-typeable
4243 blaCMY- +24 IncK2 plasmids containing a class 1 integron with aadA1, aadA2 and cmlA1
(Clone
4)
2
genes (Fig. 1).
blaSHV- -18, − 1 IncI1/ST26 The chronology of blaCMY-2 showed that the IncK2 plasmids har­
12 [tetA + (IntI1- bouring this gene were first detected in the farm in a ST973 isolate from
batch 1/S2 (two week pullets/growing house A), and two months later

6
I. Aldea et al. Veterinary Microbiology 270 (2022) 109453

in an ST1158 isolate from batch 2/S2 (two week pullets/growing house poultry meat and humans. Unfortunately, in the afore-mentioned
B). In both batches, the plasmids were also detected in the following studies, plasmid data from laying hens was only provided by Niero
sampling (S3/14 and 15 weeks pullets, respectively), but now harboured et al. (2018), who also detected blaCTX-M-1 in an IncI1 plasmid, and
by isolates belonging to clone 4 (ST4243) (Table 3). Ceccarelli et al. (2019), whose results were similar since they detected
Although this IncK2 plasmid was no longer detected in these two blaCTX-M-1 in IncI1 plasmids and blaCMY-2 in both IncI1 and IncK
batches, blaCMY-2 was detected again in batch 2/S4 (24-week hens/ plasmids.
laying house D) harboured by the IncI1/ST12 plasmid in an isolate of A ESBL blaSHV-12 gene (with a close class 1 integron gene) harboured
clone 5. This clonal isolate was also detected eight months later in the by an IncI1/ST26 plasmid in ST isolates has been previously described in
farm in batch 4/S4 in 23-week laying hens, coexisting with clone 4. E. coli isolated from broilers (Alonso et al., 2017).
In addition, the IncK2 plasmid harbouring blaCMY-2 was detected in Nevertheless, there are no previous studies reporting the presence of
three consecutive samplings from batch 3 (from day-old chicks to 14- blaSHV-12 in E. coli ST6616 isolates.
week pullets) harboured by three different ST types (ST155, 48 and
4233/clone 4) (Table 3). Therefore, the blaCMY-2 gene had been 13.1. Chromosomal location of blaCTX-M genes
spreading in the farm for at least 16 months, mainly harboured by IncK2
plasmids. Chromosomal integration of blaCTX-M-14 and blaCTX-M-15 genes has
already been described in E. coli clinical isolates (Rodriguez et al., 2014),
11. Discussion but not in isolates from poultry, such as blaCTX-M-14 in our study.
In the case of blaCTX-M-1, we found no reports of its chromosomal
When a non-selective medium was used, only one ESBL/pAmpC integration in E. coli, but only in clinical human strains of Proteus mir­
E. coli isolate was detected in this farm (Moreno et al., 2019), showing abilis (Song et al., 2011) and Enterobacter cloacae (Liu et al. (2015).
that this complex phenotype was not predominant in the farm over the In our ST93 isolates, the chromosomal gene blaCTX-M-1 was located
31 months covered by our study. Nevertheless, different isolates grew near an insertion sequence IS1380 (ISEcp1), which has also been iden­
when selective screening with 1 mg/L of CTX was performed, proving tified in the plasmid close to the same gene, suggesting it is involved in
that ESBL/pAmpC E. coli were present in the farm. A comparison be­ improving chromosomal integration of blaCTX-M-1 (Hamamoto and Hirai,
tween prevalence results using selective (CTX at 1 mg/L) and 2019). Although in our study the blaCTX-M-14 gene was not found to be
non-selective medium has been performed by Apostolakos et al. (2020b) associated with an ISECp1 element, this transposase could also facilitate
showing “no bias towards particular ESBL/pAmpC-EC genotypes from the insertion of other blaCTX-M genes.
the selective method or underestimation by the non-selective approach”.
Nevertheless, a comparison between 1 and 8 mg/L of CTX was per­ 14. Flow and persistence of ESBL/AmpC genes in the farm
formed in our laboratory (Moreno et al., 2007), showing higher preva­
lence and phenotypic diversity when using the lower CTX concentration. The detection of blaCTX-M-1 in different ST types of E. coli in a farm has
Most of the studies on ESBL/AmpC E. coli used CTX at 1 mg/L as been previously reported. Blaak et al. (2015), in a study with five laying
selective antibiotic concentration (Apostolakos et al., 2019; Chauvin hen farms, detected between four and 11 different ST E. coli isolates
et al., 2013; Dame-Korevaar et al., 2017; Dierikx et al., 2013; Wasyl harbouring blaCTX-M-1 in the same farm. Baez et al. (2021), also exam­
et al., 2012), and this is the concentration recommended by the EU ining layers, detected blaCTX-M-1 in at least 11 ST types. Among the ST
Reference laboratory for AMR (https://www.eurl-ar.eu/CustomerData/ types harbouring blaCTX-M-1 in our study, only ST10 was also detected by
Files/Folders/21-protocols/399_esbl-ampc-quantification-protocol-19 these authors.
-03-2018.pdf). Nevertheless, higher CTX concentrations, such as 2 mg/L The dynamics of ESBL/pAmpC E. coli in poultry farms during the
(Mo et al., 2014) and even 8 mg/L (Baez et al., 2021), have been also rearing period has been studied by Dierikx et al. (2013) and Baron et al.
used. Since the EUCAST epidemiological cut-off value for CTX is (2018). Dierikx et al. (2013), in a five-week longitudinal study at three
0.25 mg/L, all these techniques are unable to detect isolates with low commercial broiler farms, detected ESBL/AmpC E. coli from day-old
cefotaxime resistance levels. chick to week five in three of four batches, whereas Baron et al.
(2018), in the follow-up of several broiler flocks, detected IncI1/ST3
12. ESBL and AmpC β-lactamase genes detected plasmids harbouring blaCTX-M-1 from day two to seven (one flock) and
from day two to 41 (two flocks). Although the authors did not discuss the
All the identified genes in our study have previously been detected in clonal spread of blaCTX-M-1 in these farms, isolates from days 2 and 41
poultry (Saliu et al., 2017) and in E. coli from laying hens: blaCTX-M-1 only belonged to the same phylogenetic group in one flock, suggesting
(Baez et al., 2021; Blaak et al., 2015; Ceccarelli et al., 2019; Chauvin that blaCTX-M-1 spread in these farms was mainly plasmid-mediated. Our
et al., 2013; Niero et al., 2018; Wasyl et al., 2012), blaCTX-M-14 (Blaak results suggest longer survival periods (up to 14 months) in layers.
et al., 2015; Seo and Lee, 2019; Shim et al., 2019), blaSHV-12 (Blaak et al., In the same study, Baron et al. (2018) also detected blaCMY-2 in
2015) and blaCMY-2 (Ceccarelli et al., 2019; Chauvin et al., 2013; Seo and IncI1/ST12 plasmids from day zero to two (one flock) and from day two
Lee, 2019; Shim et al., 2019; Wasyl et al., 2012). Other ESBL genes re­ to seven (two flocks). All these isolates were of the phylogenetic group
ported in E. coli from layers are blaCTX-M-15 (Baez et al., 2021; Shim et al., A. In our study, the IncI1 plasmids harbouring blaCMY-2 were not
2019), blaLAP-2 (Baez et al., 2021) and blaTEM-52 (Blaak et al., 2015). detected in E. coli isolates from the same batch.
Dame-Korevaar et al. (2017) followed a broiler parent flock at an
13. Plasmids harbouring ESBL/AmpC genes experimental farm and detected blaCMY-2 from day 7 to week 19 during
the rearing period, but not over the laying period. blaCMY-2 persisted for
At least in one isolate on each pattern we found a plasmid replicon longer in this farm than in our study, and it was related to an IncA/C
and a ESBL/pAmpC gene in the same contig, proving that IncI1 is the type of plasmids. Curiously, the authors pointed out than an unsuc­
main platform harbouring blaCTX-M-1, blaSHV-12 and blaCMY-2. Our results cessful combination of blaCMY-2 and IncA/C plasmids was the main
confirm previous reports from poultry E. coli (reviewed by Saliu et al., reason for their inability to persist on animal level (Dame-Korevaar
2017). Similarly, the presence of blaCMY-2 in a plasmid belonging to in­ et al., 2017).
compatibility group IncK has also been previously reported in E. coli Several of the remaining studies of ESBL/pAmpC E. coli flow in the
from poultry (Agerso et al., 2014; Apostolakos et al., 2020a; Seiffert broiler production pyramid show that their vertical transmission is
et al., 2017). In fact, we detected blaCMY-2 in the IncK subgroup desig­ mainly due to plasmids, though several clues for clonal spread of blaCMY-
nated IncK2 by Seiffert et al. (2017), and detected it from poultry, 2, the most frequently detected bla gene in poultry, are also provided.

7
I. Aldea et al. Veterinary Microbiology 270 (2022) 109453

Zurfluh et al. (2014) found strong evidences that blaCTX-M-1 spread to Apostolakos, I., Mughini-Gras, L., Fasolato, L., Piccirillo, A., 2019. Assessing the
occurrence and transfer dynamics of ESBL/pAmpC-producing Escherichia coli across
the French broiler production pyramid by an IncI1/ST3 plasmid, and
the broiler production pyramid. PLoS One 14, e0217174.
Apostolakos et al. (2020a) also showed the same finding in the Italian Apostolakos, I., Feudi, C., Eichhorn, I., Palmieri, N., Fasolato, L., Schwarz, S.,
broiler production pyramid, as well as the transmission of blaSHV-12 by Piccirillo, A., 2020a. High-resolution characterisation of ESBL/pAmpC-producing
IncX3 plasmids. In our study, blaSHV-12 was harboured by IncI1/ST26 Escherichia coli isolated from the broiler production pyramid. Sci. Rep. 10, 11123.
Apostolakos, I., Mughini-Gras, L., Fasolato, L., Piccirillo, A., 2020b. Impact of selective
and a non-replicon detected plasmids. In addition, Apostolakos et al. and non-selective media on prevalence and genetic makeup of ESBL/pAmpC-
(2020a) also found IncFIB/FII plasmids harbouring blaCTX-M-55. producing Escherichia coli in the broiler production pyramid. Vet. Microbiol. 240,
Nilsson et al. (2014), using multiple-locus variable number tandem 108536.
Baez, M., Espinosa, I., Collaud, A., Miranda, I., Montano, D.L.N., Feria, A.L., Hernandez-
repeat analysis (MLVA), detected an E. coli clone carrying blaCMY-2 in the Fillor, R.E., Obregon, D., Alfonso, P., Perreten, V., 2021. Genetic Features of
Swedish broiler production pyramid, and a similar conclusion about the Extended-Spectrum beta-Lactamase-Producing Escherichia coli from Poultry in
transmission of this bla gene in the Danish pyramid was reached by Mayabeque Province, Cuba. Antibiot. (Basel) 10.
Baron, S., Le L., Devendec, Touzain, F., Jouy, E., Lucas, P., de Boisseson, C., Larvor, E.,
Agerso et al. (2014) using pulsed-field electrophoresis (PFGE); they also Kempf, I., 2018. Longitudinal study of Escherichia coli plasmid resistance to
detected blaCMY-2 in IncI1 and IncK plasmids. This Russian doll model extended-spectrum cephalosporins in free-range broilers. Vet. Microbiol. 216,
(blaCMY-2 inside an IncK plasmid inside a clonal E. coli strain) was also 20–24.
Blaak, H., van Hoek, A.H., Hamidjaja, R.A., van der Plaats, R.Q., Kerkhof-de Heer, L., de
detected by Apostolakos et al. (2020a) in the Italian broiler production Roda Husman, A.M., Schets, F.M., 2015. Distribution, numbers, and diversity of
pyramid. ESBL-producing E. coli in the poultry farm environment. PLoS One 10, e0135402.
Although the source of the ESBL/pAmpC E. coli in the studied farm Ceccarelli, D., Kant, A., van Essen-Zandbergen, A., Dierikx, C., Hordijk, J., Wit, B.,
Mevius, D.J., Veldman, K.T., 2019. Diversity of plasmids and genes encoding
was not examined, their isolation from day-old chick in one of the four
resistance to extended spectrum cephalosporins in commensal escherichia coli from
batches studied suggested that an external source must be consider. dutch livestock in 2007-2017. Front Microbiol 10, 76.
Nevertheless it is impossible to rule out environmental contamination, Chauvin, C., Le L., Devendec, Jouy, E., Le Cornec, M., Francart, S., Marois-Crehan, C.,
as detected in other studies (Dame-Korevaar et al., 2017; Dierikx et al., Kempf, I., 2013. National prevalence of resistance to third-generation cephalosporins
in Escherichia coli isolates from layer flocks in France. Antimicrob. Agents
2013) as a reason for their persistence in the farm. Chemother. 57, 6351–6353.
According to this and other studies, it is clear that, although anti­ Curiao, T., Canton, R., Garcillan-Barcia, M.P., de la Cruz, F., Baquero, F., Coque, T.M.,
microbial use is a well-known driver of AMR, in the case of commercial 2011. Association of composite IS26-sul3 elements with highly transmissible IncI1
plasmids in extended-spectrum-beta-lactamase-producing Escherichia coli clones
farms of both layers and broilers it does not appear to be the only factor from humans. Antimicrob. Agents Chemother. 55, 2451–2457.
responsible for the persistence of ESBL/pAmpC E. coli at farm level. Dame-Korevaar, A., Fischer, E.A.J., Stegeman, A., Mevius, D., van Essen-Zandbergen, A.,
Therefore, other farm management strategies in addition to proper Velkers, F., van der Goot, J., 2017. Dynamics of CMY-2 producing E. coli in a broiler
parent flock. Vet. Microbiol 203, 211–214.
antimicrobial use should be implemented to control this spread. Dierikx, C.M., van der Goot, J.A., Smith, H.E., Kant, A., Mevius, D.J., 2013. Presence of
ESBL/AmpC-producing Escherichia coli in the broiler production pyramid: a
descriptive study. PLoS One 8, e79005.
Funding Galardini, M., Biondi, E.G., Bazzicalupo, M., Mengoni, A., 2011. CONTIGuator: a
bacterial genomes finishing tool for structural insights on draft genomes. Source
This work was funded by a grant from the Ministerio de Economía y Code Biol. Med. 6, 11.
Grande Burgos, M.J., Fernandez Marquez, M.L., Perez Pulido, R., Galvez, A., Lucas
Competitividad (grant number RTA2014–00012-C03–03). IA received
Lopez, R., 2016. Virulence factors and antimicrobial resistance in Escherichia coli
funding from the European Union’s Horizon 2020 Research and Inno­ strains isolated from hen egg shells. Int J. Food Microbiol. 238, 89–95.
vation programme, under grant agreement No 773830: One Health Hamamoto, K., Hirai, I., 2019. Characterisation of chromosomally-located blaCTX-M and
European Joint Programme. its surrounding sequence in CTX-M-type extended-spectrum beta-lactamase-
producing Escherichia coli isolates. J. Glob. Antimicrob. Resist. 17, 53–57.
Kaas, R.S., Leekitcharoenphon, P., Aarestrup, F.M., Lund, O., 2014. Solving the problem
of comparing whole bacterial genomes across different sequencing platforms. PLoS
Declaration of Competing Interest One 9, e104984.
Letunic, I., Bork, P., 2019. Interactive Tree Of Life (iTOL) v4: recent updates and new
developments. Nucleic Acids Res. 47, W256–W259.
The authors declare that they have no known competing financial Liu, J., Zeng, T., Su, G., Lin, L.Y., Zhao, Y., Yang, W.Q., Xie, W.X., Zhao, Z.G., Li, G.M.,
interests or personal relationships that could have appeared to influence 2015. The dissemination mode of drug-resistant genes in Enterobacter cloacae.
Indian J. Med Microbiol. 33, 87–92.
the work reported in this paper. Mo, S.S., Norstrom, M., Slettemeas, J.S., Lovland, A., Urdahl, A.M., Sunde, M., 2014.
Emergence of AmpC-producing Escherichia coli in the broiler production chain in a
country with a low antimicrobial usage profile. Vet. Microbiol. 171, 315–320.
Acknowledgements Moreno, M.A., Teshager, T., Porrero, M.C., Garcia, M., Escudero, E., Torres, C.,
Dominguez, L., 2007. Abundance and phenotypic diversity of Escherichia coli
The authors are indebted to the owners of the farm where this study isolates with diminished susceptibility to expanded-spectrum cephalosporins in
faeces from healthy food animals after slaughter. Vet. Microbiol. 120, 363–369.
was performed. The authors also wish to thank the technicians María
Moreno, M.A., Garcia-Soto, S., Hernandez, M., Barcena, C., Rodriguez-Lazaro, D., Ugarte-
García, Estefanía Rivero, Nisrin Maasoumi, Celia Fernández and Este­ Ruiz, M., Dominguez, L., 2019. Day-old chicks are a source of antimicrobial resistant
fanía Fernández for their excellent technical assistance at the VISAVET bacteria for laying hen farms. Vet. Microbiol. 230, 221–227.
Niero, G., Bortolaia, V., Vanni, M., Intorre, L., Guardabassi, L., Piccirillo, A., 2018. High
Foodborne Zoonoses and Antibiotic Resistance Unit.
diversity of genes and plasmids encoding resistance to third-generation
cephalosporins and quinolones in clinical Escherichia coli from commercial poultry
Appendix A. Supporting information flocks in Italy. Vet. Microbiol. 216, 93–98.
Nilsson, O., Borjesson, S., Landen, A., Bengtsson, B., 2014. Vertical transmission of
Escherichia coli carrying plasmid-mediated AmpC (pAmpC) through the broiler
Supplementary data associated with this article can be found in the production pyramid. J. Antimicrob. Chemother. 69, 1497–1500.
online version at doi:10.1016/j.vetmic.2022.109453. Rasheed, M.U., Thajuddin, N., Ahamed, P., Teklemariam, Z., Jamil, K., 2014.
Antimicrobial drug resistance in strains of Escherichia coli isolated from food
sources. Rev. Inst. Med Trop. Sao Paulo 56, 341–346.
References Rodriguez, I., Thomas, K., Van Essen, A., Schink, A.K., Day, M., Chattaway, M., Wu, G.,
Mevius, D., Helmuth, R., Guerra, B., Consortium, S.-E., 2014. Chromosomal location
of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The
Agerso, Y., Jensen, J.D., Hasman, H., Pedersen, K., 2014. Spread of extended spectrum
Netherlands and the UK. Int. J. Antimicrob. Agents 43, 553–557.
cephalosporinase-producing Escherichia coli clones and plasmids from parent
Roer, L., Overballe-Petersen, S., Hansen, F., Johannesen, T.B., Stegger, M., Bortolaia, V.,
animals to broilers and to broiler meat in a production without use of
Leekitcharoenphon, P., Korsgaard, H.B., Seyfarth, A.M., Mossong, J., Wattiau, P.,
cephalosporins. Foodborne Pathog. Dis. 11, 740–746.
Boland, C., Hansen, D.S., Hasman, H., Hammerum, A.M., Hendriksen, R.S., 2019.
Alonso, C.A., Michael, G.B., Li, J., Somalo, S., Simon, C., Wang, Y., Kaspar, H., Kadlec, K.,
ST131 fimH22 Escherichia coli isolate with a blaCMY-2/IncI1/ST12 plasmid
Torres, C., Schwarz, S., 2017. Analysis of blaSHV-12-carrying Escherichia coli clones
obtained from a patient with bloodstream infection: highly similar to E. coli isolates
and plasmids from human, animal and food sources. J. Antimicrob. Chemother. 72,
of broiler origin. J. Antimicrob. Chemother. 74, 557–560.
1589–1596.

8
I. Aldea et al. Veterinary Microbiology 270 (2022) 109453

Saliu, E.M., Vahjen, W., Zentek, J., 2017. Types and prevalence of extended-spectrum spectrum beta-lactamases in clinical isolates of Proteus mirabilis from Korea.
beta-lactamase producing Enterobacteriaceae in poultry. Anim. Health Res Rev. 18, Antimicrob. Agents Chemother. 55, 1414–1419.
46–57. Stewardson, A.J., Renzi, G., Maury, N., Vaudaux, C., Brossier, C., Fritsch, E., Pittet, D.,
Seiffert, S.N., Carattoli, A., Schwendener, S., Collaud, A., Endimiani, A., Perreten, V., Heck, M., van der Zwaluw, K., Reuland, E.A., van de Laar, T., Snelders, E.,
2017. Plasmids carrying blaCMY -2/4 in escherichia coli from poultry, poultry meat, Vandenbroucke-Grauls, C., Kluytmans, J., Edder, P., Schrenzel, J., Harbarth, S.,
and humans belong to a novel IncK subgroup designated IncK2. Front. Microbiol. 8, 2014. Extended-spectrum beta-lactamase-producing Enterobacteriaceae in hospital
407. food: a risk assessment. Infect. Control Hosp. Epidemiol. 35, 375–383.
Seo, K.W., Lee, Y.J., 2019. Detection of plasmid-mediated quinolone resistance genes in Wasyl, D., Hasman, H., Cavaco, L.M., Aarestrup, F.M., 2012. Prevalence and
beta-lactamase-producing Escherichia coli isolates from layer hens. Poult. Sci. 98, characterization of cephalosporin resistance in nonpathogenic Escherichia coli from
1480–1487. food-producing animals slaughtered in Poland. Micro Drug Resist. 18, 79–82.
Shim, J.B., Seo, K.W., Kim, Y.B., Jeon, H.Y., Lim, S.K., Lee, Y.J., 2019. Molecular Zurfluh, K., Wang, J., Klumpp, J., Nuesch-Inderbinen, M., Fanning, S., Stephan, R., 2014.
characteristics of extended-spectrum and plasmid-mediated AmpC beta-lactamase- Vertical transmission of highly similar bla CTX-M-1-harboring IncI1 plasmids in
producing Escherichia coli isolated from commercial layer in Korea. Poult. Sci. 98, Escherichia coli with different MLST types in the poultry production pyramid. Front.
949–956. Microbiol. 5, 519.
Song, W., Kim, J., Bae, I.K., Jeong, S.H., Seo, Y.H., Shin, J.H., Jang, S.J., Uh, Y., Shin, J.
H., Lee, M.K., Lee, K., 2011. Chromosome-encoded AmpC and CTX-M extended-