https://doi.org/10.52973/rcfcv-e33257
Received: 11/04/2023 Accepted: 04/05/2023 Published: 15/05/2023
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Revista Científica, FCV-LUZ / Vol. XXXIII, rcfcv-e33257, 1 – 7
ABSTRACT
Systemic infections by avian pathogenic Escherichia coli (APEC)
are economically damaging to poultry industries Worldwide. E. coli
strains of serotypes O1, O2, O18 and O78 are preferentially associated
with avian colibacillosis. The rfb gene cluster that controls O antigen
synthesis generally varies among different E. coli serotypes. In this
study, the rfb gene clusters of E. coli serotypes O1, O2, O18 and O78
were characterized and compared, and it was also aimed to search for
Colistin resistance on a molecular basis. For the research, 200 swab
samples were taken from 200 chickens suspected of colibacillosis in
broiler poultry farms located in the vicinity of Aydın, İzmir, and Manisa
Provinces in Turkey 2022. Bacterial growth was obtained from 92%
of the samples, and microbiological analysis identied 108 (54%)
Escherichia coli isolates. In addition, Klebsiella spp. was identied in
35 (17.5%) samples, Proteus spp. in 23 (11.5%), Pseudomonas spp. in 18
(9%), and no bacterial growth was observed in 16 (8%) samples. mcr-1
(309 bp) and mcr–2 (567 bp) genes responsible for Colistin resistance
was investigated in plasmid DNA extracted from 108 E. coli isolates
obtained in the study, using the PCR method. However, neither mcr-1
nor mcr–2 genes were detected in any of the samples. In conclusion,
the allele-specic PCR method was found sensitive and applicable
for APEC identication and multiple drug resistance emerged in E.
coli strains isolated according to the antibiogram results.
Key words: Avian pathogenic Escherichia coli; mcr gene; antibiotic
resistance
RESUMEN
Las infecciones sistémicas por Escherichia coli patógena aviar (APEC)
son económicamente dañinas para las industrias avícolas en todo
el mundo. Las cepas de E. coli de los serotipos O1, O2, O18 y O78 se
asocian preferentemente con la colibacilosis aviar. El grupo de genes
rfb que controla la síntesis del antígeno O generalmente varía entre los
diferentes serotipos de E. coli. En este estudio, los grupos de genes
rfb de los serotipos O1, O2, O18 y O78 de E. coli se caracterizaron y
compararon, y también tuvo como objetivo buscar la resistencia a la
Colistina sobre una base molecular. Para la investigación, se tomaron
muestras de hisopados de 200 pollos con sospecha de colibacilosis
en las granjas avícolas de pollos de engorde en las provincias de
Aydın, İzmir y Manisa, en Turkia, 2022. Como resultado del análisis
microbiológico de las muestras se identicaron 108 (54 %) Escherichia
coli. Además, Klebsiella spp. en 35 (17,5 %) de las muestras, Proteus
spp., en 23 (11,5 %) y Pseudomonas spp., en 18 (9 %) de las muestras
identicadas y el crecimiento bacteriano no ocurrió en 16 (8 %) de
ellos. La presencia de los genes mcr-1 (309 pb) y mcr–2 (567 pb)
responsables de la resistencia a la Colistina en los ADN plasmídicos
extraídos de 108 aislados de E. coli obtenidos en el estudio, se
investigó mediante el método PCR. Como resultado de la amplicación
por PCR, los genes mcr-1 y mcr–2 no pudieron detectarse en ninguna
de las muestras. El método de PCR se evaluó como un método sensible
y aplicable para la identicación y serotipicación de APEC, y se
determinó el perl de resistencia a múltiples fármacos en las cepas
de E. coli aisladas de acuerdo con los resultados del antibiograma.
Palabras clave: Escherichia coli patógena aviar; gen mcr; resistencia
a antibióticos
Serotyping of Escherichia coli species isolated from broilers and
determination of Colistin resistance
Serotipado de Escherichia coli aisladas de pollos de engorde y determinación de resistencia a la
Colistina
Ugur Parin
1
* , Gonenc Simsek
2
1
Aydin Adnan Menderes University, Faculty of Veterinary Medicine, Department of Microbiology Isikli. Aydin, Turkey.
2
Aydin Adnan Menderes University, Institute of Health Sciences, Department of Microbiology Efeler. Aydin, Turkey.
*Corresponding author: uparin@adu.edu.tr
Molecular characterization of APEC isolated from broilers / Parin and Simsek ______________________________________________________
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INTRODUCTION
Escherichia coli is one of the most important pathogens causing
enteritis and septicaemia in various species such as poultry (Gallus
gallus domesticus), pigs (Sus scrofa), ruminants (Bos taurus), dogs
(Canis lupus familiaris), cats (Felis catus), horses (Equus caballus)
and rabbits (Oryctolagus cuniculus). E. coli, which causes local
or systemic infections in poultry, was first isolated by German
paediatrician Theodor Escherich and named Escherichia to honour
him. Avian pathogenic E. coli (APEC) is the cause of colisepticaemia,
coligranuloma (Hjarre's disease), air sacculitis, coliform cellulitis,
bulging head syndrome, coliform peritonitis, coliform salpingitis,
coliform osteomyelitis/synovitis, coliform panophthalmitis and
coliform cheocephalitis [1]. It is known to cause inammation (yolk
sac infection) and colibacillosis is one of the most commonly observed
diseases among poultry diseases of bacterial origin. In the 1990s,
deaths due to septicaemia were among the most common cases
in laying hen farms in Europe. In addition, APEC-related infections
in layers and broiler chickens were reported in Belgium between
1997 and 2000 were 17.7 and 38.6%, respectively, and resistance to
antibiotics was also high [2, 3].
Serotyping of E. coli is due to the O antigen in lipopolysaccharide
and the H antigen in agella. O antigen denotes serogroup and H
antigen denotes serotype. Capsular antigen (K) is also used for
classication. Somatic O antigens are heat-resistant surface antigens
in lipopolysaccharide structure. Lipopolysaccharide, also known as
endotoxins, are found on the outer membrane of Gram-negative
bacilli cell wall. Somatic O antigens Although they are important in
the serogrouping of E. coli, and they can be revealed by agglutination
test. Flagellar H antigen varies according to the different types of
agellin protein found in the structure of the agella. H antigen,
which is heat labile, can be detected by agglutination test. Capsular
K antigen is in polymeric acid structure. They are present on the
cell surface and prevent the agglutination of the O antigen. They are
divided into L, A and B subclasses according to their heat sensitivity.
Fimbrial (Pilus) antigens were previously called the L subclass of K
antigens, but as a result of the studies, currently is well known they
are located in the mbriae of E. coli. The pilus, which plays a role in
the adhesion of bacteria to intestinal epithelial cells, is divided into
mannose-sensitive and mannose-resistant [2].
Generally, O1, O2, O8, O15, O18, O35, O78, O88, O109 and O115
serogroups of O antigen have been detected in colibacillosis
infections of poultry. O1, O2 and O78 were the most prevalently
detected serogroups. Pathogenic E. coli species are divided into
intestinal and extraintestinal E. coli pathotypes according to the type
of infection. Intestinal pathogenic E. coli (IPEC) causing diarrhoea
are enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC),
enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC) and
enteroaggregative E. coli (EAEC). Extraintestinal pathogenic E. coli
species are divided into uropathogenic E. coli (UPEC), septicemia-
causing E. coli (SePEC), newborn meningitis-causing E. coli (NMEC)
and avian pathogenic E. coli (APEC) [4].
The scope of the research was to analyse the rfb gene groups of
the dominant serovars of avian pathogenic E. coli, including O1, O2,
O18, and O78 strains, and to determine the serotyping of O-antigens
with an allele-specic Polymerase chain reaction (PCR) method. The
sensitivity and applicability of the allele-specic PCR method for
APEC identication and serotyping were investigated, and Colistin
resistance was searched by molecular methods.
MATERIAL AND METHODS
Animal specimen, isolation methods and primers
For the research, cloacal swab samples were taken from 200
chickens with suspected colibacillosis and natural death from broiler
poultry houses around Aydın, İzmir and Manisa Provinces in Turkey
2022. The samples were brought to Adnan Menderes University,
Faculty of Veterinary Medicine, Department of Microbiology, Routine
Diagnostic Laboratory in cold chain. Swap samples were incubated
at 37°C for 24 h after inoculation on MacConkey agar. Lactose
positive pink colonies were seeded on EMB agar and incubated at
37°C overnight. Colonies that gave metallic green sheen on EMB
medium after incubation were puried, and E. coli was identied by
Gram staining, indole, methyl red, Voges-Proskauer, citrate catalase,
oxidase reactions and motility examinations after purification.
Serotyping with various commercial antisera kits (O1, O2, O5, O6, O8,
O9, O11, O12, O14, O15, O17, O18, O20, O35, O36, O45, O53, O78, O81, O83,
O88, O102, O103, O115, O116 and O132) (Mast Assure™, UK) was applied
to the isolates identied as E. coli. O1, O2, O18 and O78 serotypes
identied after serotyping according to agglutination reactions were
conrmed by allele-specic PCR method. Serotyping isolates and
other identied E. coli strains were PCR processed with mcr–1 and
mcr–2 specic primers to determine Colistin resistance. The primer
sequences used in the study are shown in TABLE I below [5, 6, 7].
TABLE I
The primer sequences used in the study
Target
gene
Primer Sequences (5'-3')
Fragment
lenght
Reference
ECO-F
(gnd)
F: 5'-CGATGTTGAGCGCAAGGTTG-3'
[5]
ECO1-R
(rfbO1)
R:5'-CATTAGGTGTCTCTGGCACG-3' 263 bp
[5]
ECO2-R
(rfbO2)
sR:5'-GATAAGGAATGCACATCGCC-3' 355 bp
[5]
ECO18-R
(rfbO18)
R:5'-AGAAGCATTGAGCTGTGGAC-3' 459 bp
[5]
ECO78-R
(rfbO78)
R: 5'-TAGGTATTCCTGTTGCGGAG-3' 623 bp
[5]
mcr-1
CLR F: 5'-CGGTCAGTCCGTTTGTTC-3'
309 bp
[6]
CLR R: 5'-CTTGGTCGGTCTGTAGGG-3'
mcr–2
MCR2 IF: 5' – TGTTGCTTGTGCCGATTGGA-3'
567 bp
[7]
MCR2 IR: 5'-AGATGGTATTGTTGGTTGCTG-3'
Genomic DNA isolation
The deoxyribonucleic acid (DNA) was extracted from pure E. coli
isolates using a bacterial DNA isolation kit (Fermentas, Lithuania),
according to the manufacturer’s instructions. All isolation steps were
completed in 1.5 mL microcentrifuge tubes.
PCR
In allele-specic PCR for serotyping, the conditions were initial
denaturation at 95°C for 5 min, followed by 30 cycles of 35 s
denaturation, at 95°C, 30 s annealing at 57°C, 40 s extension at 72°C
and, 10 min of nal extension at 72°C. For the PCR mix, in a total
FIGURE 1. PCR gel electrophoresis image of serotypes Gel size of E. coli serovars
O1, O2, O18 and O78. M: DL2000 DNA Marker; O1, O2, O18 and O78 represent PCR
products for serovars O1, O2, O18 and O78, respectively. 1: Positive control APEC
IMT2467; 2: Isolate APEC O1; 3: Positive control APEC IMT555; 4: Isolate APEC O2;
5: Positive control APEC Field strain; 6: Isolate APEC O18; 7: Positive control APEC
IMT663; 8: Isolate APEC O78; 9: Negative control (P. aeruginosa ATCC 27853)
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volume of 25 µL, 2.5 µL of 10X PCR Buffer (25mM MgCl
2
), 2 µL of dNTP
(2.5mM of each dNTP), 1.5 U of Taq polymerase, 0.5 µL of each primer
(10 µM), and 1 µL of DNA sample was used [5].
In the PCR procedure to determine Colistin resistance, the conditions
were initial denaturation at 94°C for 15 min, followed by 25 cycles of
denaturation at 94°C for 30 s, annealing at 58°C for 90 s, extension at
72°C for 60 s and, consisted of a 10 min nal extension phase at 72°C.
For the PCR mix, 8.5 µL of milliQ water, 12.5 µL of 2X PCR mix, 0.5 µL of
each primer (2 µM) and 2 µL of DNA sample were used in a total volume
of 25 µL. PCR products were analysed by agarose gel electrophoresis
(Agarose-ME, Classic Type; Nacalai Tesque, Inc., Japan), and visualized
in a UV device (Innity VX2, France) . Target electrophoretic bands
with specic fragment length were visualized (Mastercycler Personal;
Eppendorf, Netheler, Hinz GmbH, Hamburg, Germany). E. coli IMT 2467
(serotype O1), E. coli IMT 5155 (serotype O2), E. coli IMT 663 (serotype
O78) were used as positive controls. All these positive controls for the
specic genes were obtained from of AniCon Labor GmbH (AniCon
Labor GmbH, Emstek, Germany). The positive control strains of Colistin
resistance were obtained from NCTC as E. coli 14377 (mcr–1) and E. coli
14378 (mcr–2). For the positive control of serotype O18, eld strain of
department laboratory isolated from chicken were used.
Antibiotic susceptibility test
Antibiotic susceptibility test of the isolates was performed following
disk diffusion method as recommended by Clinical and Laboratory
Standards Institute (CLSI) [8]. A loopful of pure cultures of the identied
agents was taken and inoculated into 5 mL of Brain-Heart Infusion
broth, and the media were incubated for 24 h at 37°C. The bacterial
suspension obtained was adjusted to 0.5 McFarland turbidity and 100 µL
was taken and after inoculation on Mueller-Hinton agar, antibiotic discs
were placed on the agar at appropriate intervals. After 24 h of incubation
(Nüve EN500, Turkey) of the media at 37°C, the zone diameters
around the antibiotic discs were measured in millimetric scale and
the antibiotic susceptibility of bacterial agents was determined. The
antimicrobial agents used in this research were as follows: amikacin
(AK), Ampicillin (AMP), Colistin (CL), Cotrimoxazole (SXT), Ciprooxacin
(CIP), Doxycycline (DO), gentamicin (GEN), levofloxacin (LE) and
nitrofurantoin (NIT). For quality control of antibiotic susceptibility
test, E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853
were used as reference strains [9].
RESULTS AND DISCUSSION
Isolation and identication ndings of the serotypes
In this study, E. coli was identied from 108 (54%) of 200 cloacal swab
samples. In addition, Klebsiella spp. from 35 (17.5%) of the samples,
Proteus spp. from 23 (11.5%) and Pseudomonas spp. from 18 (9%) of the
samples. Gram negative bacterial growth did not occur in 16 (8%) of
them. Bacteria isolated and identied from cloacal swabs from broiler
chickens are shown in TABLE II. The distribution of the identied E. coli
isolates according to O1, O2, O18, and O78 serotypes is shown in TABLE
III. The distribution of isolated serotypes by cities is shown in TABLE IV.
PCR ndings
PCR gel electrophoresis image of serotypes is given in FIG. 1.
In order to determine the genotypic resistance, 108 isolates were
extracted by the previously mentioned methods. All of 9 APEC O1,
11 APEC O2, 6 APEC O18 and 39 APEC O78 serotypes isolated in this
TABLE II
Bacteria isolated and identied from cloacal
swabs from broiler chickens
Bacterial species Isolate number (%)
E. coli spp. 108 54
Klebsiella spp. 35 17.5
Proteus spp. 23 11.5
Pseudomonas spp. 18 9
No bacterial growth 16 8
TOTAL 200 100.00
TABLE III
The distribution of the identied E. coli isolates
according to O1, O2, O18, and O78 serotypes
Serotypes
APEC isolates (n=108)
PCR Serum agglutination
O1 9 8
O2 11 10
O18 6 6
O78 39 36
O1, O2, O18, O78 0 5
Other serotypes – 43
TABLE IV
The distribution of isolated serotypes by cities
Cities Sample
E. coli
O1
E. coli
O2
E. coli
O18
E. coli
O78
Other
Manisa 70 5 6 3 17 16
İzmir 70 3 3 3 12 14
Aydın 60 1 2 – 10 13
TOTAL 200 9 11 6 39 43
FIGURE 2. Resistance gene PCR image M: Marker 1 and 2: Positive controls (E.
coli 14377 and 14378) 3: Negative control (P. aeruginosa ATCC 27853) 4-9: Isolates
studied in this research
Molecular characterization of APEC isolated from broilers / Parin and Simsek ______________________________________________________
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study were conrmed by allele specic PCR test. The results indicated
that 65 (60%) of 108 E. coli isolates showed corresponding PCR bands
at respective size, which was in accordance with bacteriological
examination and conventional serum agglutination assay. The
presence of mcr-1 (309 bp) and mcr–2 (567 bp) genes responsible for
Colistin resistance in plasmid DNAs extracted from 108 E. coli isolates
obtained in the study were investigated by PCR method. As a result
of PCR amplication, mcr-1 and mcr–2 genes could not be detected
in any of the samples. Resistance gene PCR image is given in FIG. 2.
Antibiotic susceptibility ndings
None of the nine antibiotics tested introduced 100% ecacy versus
E. coli strains. According to the antibiogram results, 98% of strains
were resistant to Ampicillin at the highest rate and 16% of strains were
resistant to Amikacin at the lowest rate. Cotrimoxazole, Doxycycline,
and Ciprooxacin were found to be responsible for more than 60%
resistance. In addition, 5 (7.6%) of 65 isolates were phenotypically
resistant to Colistin.
One of the leading causes of disease and mortality in chickens
across the Globe is colibacillosis. The majority of instances of avian
colibacillosis are caused by APEC serotypes O1, O2, O18, and O78
strains. In some APEC outbreaks, many strains with varied serotypes
have been discovered. Additionally, trans protection among several
APEC serovars is weak [10]. Thus, for APEC control, a quick and
precise serotyping approach is essential. Conventional APEC
serotyping methods like the serum agglutination test require a
spectrum of strong antisera versus bacterial isolates and several
O-antigens in order to produce results. Serum agglutination cannot
differentiate APEC serotypes in a single test. Furthermore, a strain
may react with more than one APEC antiserum. Hence, an allele-
specic PCR test was applied for serotyping the dominant APEC
serotypes in this research. Between the rfb locus and the gnd
gene, distinct O-antigen sequences were amplied using PCR in
various sizes. This study showed that the PCR test can be applied
to serotyping APEC O1, O2, O18, and O78 strains in bacterial cultures
and did not react with reference strains of E. coli and other bacterial
species with different serotypes. Furthermore, multiple agglutination
isolates can be clearly serotyped using this method, indicating that
the PCR test is specic and reliable.
The PCR test was successful in serotyping APEC strains in clinical
infected tissue samples, suggesting its use for laboratory detection.
Additionally, it was able to distinguish serotypes of multi-agglutination
samples in routine bacteriological examination. Therefore, this
PCR test was found to be more accurate than conventional serum
agglutination assays. The APEC clinical diagnosis and epidemiological
research were made easier with lighter effort and in a short amount
of period. To summarize, the study utilized an allele-specific
PCR test that had the ability to accurately differentiate between
the dominant APEC serotypes (O1, O2, O18, and O78) with great
precision and sensitivity. This approach addresses the limitations
of traditional serological tests and offers an ecient and suitable
method for identifying the predominant APEC strains. Consequently,
using this PCR test offers advantages for clinical diagnosis and
epidemiological surveys.
One of the most common mechanismsis the acquisitionof
resistance genes through horizontal gene transfer. This process
involves the transfer of genetic material from one bacterium to
another, often facilitated by plasmids or other mobile genetic
elements.Another mechanism of antibiotic resistance in APEC is
the presence of eux pumps. These pumps are proteins that are
able to remove antibiotics from the bacterial cell before they can
exert their effects. This mechanism of resistance is particularly
common in Fluoroquinolone – resistant APEC strains. Studies have
shown that antibiotic resistance is a growing problem in APEC
infections. Resistance to Fluoroquinolones and Tetracyclines is
particularly common, with some APEC strains showing resistance
to multiple antibiotics. In some cases, resistance to one antibiotic
can also confer cross-resistance to other antibiotics. For example,
resistance to Tetracyclines can often lead to resistance to other
antibiotics in the same class, such as Doxycycline and Minocycline.
The prevalence of antibiotic resistance in APEC strains varies
depending on geographical location and other factors. Antibiotics
are widely used in the commercial poultry sector to treat illnesses
and encourage growth [11]. Globally, it is estimated that antimicrobial
consumption in animal food production will increase by 67% by 2030.
In the Asia-Pacic Region, the use of antimicrobials in chickens is
expected to increase by 129% by 2030. Avian colibacillosis is the
leading disease reported in chickens in several previous studies.
The use of antibacterial as a treatment approach in the chicken
industry is successful in reducing the danger of colibacillosis, but the
introduction of multi-drug resistant bacteria and the transmission
of resistance genes have made this task much more dicult [12].
None of the nine tested antibiotics introduced 100% ecacy versus
E. coli strains. Ampicillin showed the highest resistance rate among
E. coli isolates, with up to 98% being resistant to it. On the other
hand, the lowest resistance rate was observed for Amikacin, with
only 16% of the isolates showing resistance to it. Trimethoprim-
Sulfamethoxazole, Doxycycline and Ciprooxacin were responsible for
more than 60% of the resistance. E. coli serovar patterns of antibiotic
resistance were similar to those documented in earlier research
[13]. On the other hand, Amikacin was the most successful against
84% of strains, as demonstrated by Bist et al. [14]. The improper
implementation of various drugs in chicken feed and treatment is
a prevalent application [15]. The indiscriminate use of antibiotics
applies a selection pressure that leads to the development of drug-
resistant bacterial strains. The patterns of antibiotic resistance
discovered in this study point to a concerning incidence of resistant
E. coli serovars in broiler chickens in the Aegean Region of Turkey.
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Multi-antibiotic resistance patterns showed that 94% of the isolates
were resistant to three or more antimicrobials. The high prevalence
of multidrug resistance (MDR) in E. coli has been documented in
Bangladesh, China, and Korea [16, 17]. The range of serovars with an
MAR index higher than 0.2 was 94%, while the proportion of isolates
with an MAR index of 0.2 or less was 6%. An MAR index value greater
than 0.2 indicates high-risk contamination sources, where various
antibiotics can frequently be used to control diseases [18]. This is
a strong indicator of the indiscriminate and improper use of drugs.
Antibiotic-resistant microbes eventually take the place of antibiotic-
sensitive ones in the environment [19].
Colistin is used as a last resort in bacteria that show multiple
resistances. Its use has been restricted in many countries due to
its negative effects on human health over time. However, currently,
the Colistin antibiotic is extensively applied and marketed in chicken
breeding. Yet, it is also referred to as a saviour for diseases brought
on by germs that are resistant to antibiotics, which is one of the
most significant current challenges [20]. The mcr-1 and mcr–2
are genes found in E. coli that encode enzymes known as Colistin
resistance proteins. These genes encode enzymes known as
phosphoethanolamine transferases, which modify a component of
the bacterial cell membrane called lipid A. This modication reduces
the annealing of Colistin to the cell membrane, thereby reducing its
effectiveness as an antibiotic. The mcr-1 and mcr–2 genes are part of
a larger family of genes called the mobilized Colistin resistance (mcr)
genes. In addition, there is evidence to suggest that the mcr-1 gene
can be transferred between different bacterial species, including
those that are normally not considered to be pathogenic. This raises
concerns about the potential for the emergence of new pathogenic
bacteria that are resistant to multiple antibiotics. Bacterial resistance
to different antibiotics is developing quickly, and worrisomely, Colistin
has recently begun to appear on this list of medications. Although
there have not been many instances of Colistin resistance in Turkey
yet, it is thought to be a potential emergence in the upcoming years.
Bacteria develop resistance to antibiotics through unique systems
and mechanisms, and then spread them [21]. Hence, it is important
to conduct studies targeted at detecting Colistin resistance, building
a Worldwide and local gene pool for it, and guring out how common
the essential gene is.
For genomic characterization of Colistin resistance, PCR techniques
are frequently used. Spectrouorometry (Lumina, Thermo Fischer
Scientic, USA), MALDI-TOF MS, microarray, and multiplex real-
time PCR methods are future detection methods predicted to be
used [22]. However, Irrgang et al. [23] suggested that the newly
developed TaqMan-based real-time PCR could effectively and quickly
detect the mcr-1 gene. Liu et al. [6] reported the rst occurrence of
plasmid-mediated Colistin resistance with the mcr-1 gene in E. coli
and Klebsiella spp. The mcr-1 gene was found in 14.9% of commercially
available pork and chicken meats, 20.6% of their samples from pigs,
and 1.4% of their isolates from patients in China during the identical
study. Research reported afterwards in many nations have revealed
that the mcr-1 gene is becoming more widespread globally [7, 24, 25,
26, 27, 28, 29, 30, 31, 32].
In this study, resistance was observed in 5 of the 65 isolates
examined (7.6%) phenotypically. The recorded rate can be regarded
as noteworthy even though it is in accordance with published data.
The fth-most utilized veterinary antibiotic in the European Union,
according to reports, is Colistin [33]. Colistin is prevalently applied
in Turkey. This circumstance can be interpreted as the cause of the
reported rate in our investigation. The lack of the pertinent genes
encoding (mcr-1 and 2), meanwhile, raises the possibility that the
resistance is chromosomal or that there are additional genes that
contribute to the resistance.
Corresponding to this, researchers in Iran examined at the Colistin
resistance-causing mcr-1, mcr–2, and plasmid encoded crrB genes.
The research examined nine hundred bacterial isolates, and 3.33% of
them showed phenotypic Colistin resistance. However, no plasmid-
mediated resistance-providing mcr-1 and mcr–2 genes were found
in any of the samples studied. The authors agreed that the phoP and
phoQ genes on the chromosome is possibly to reason for the Colistin
resistance observed in Northwest Iran. APEC strains isolated from
turkeys showed high levels of resistance to Fluoroquinolones and
Tetracyclines. Antibiotic resistance in APEC can have signicant
consequences for the health of poultry and the economic viability of
the poultry industry. Infections caused by antibiotic-resistant APEC
strains are often more dicult to treat, leading to higher morbidity
and mortality rates in affected birds [13, 34].
This, in turn, can lead to signicant economic losses for poultry
producers. In addition to the impact on animal health, antibiotic-
resistant APEC strains also pose a potential risk to human health.
There is evidence to suggest that APEC strains can be transmitted
from poultry to humans, and that these strains may carry resistance
genes that can make infections dicult to treat. In some cases,
infections caused by antibiotic-resistant APEC strains have been
associated with increased morbidity and mortality in humans [34].
Recently, conducted studies have shown that the mcr–2, mcr–3,
mcr–4, mcr–5, mcr–6, mcr–7, mcr–7.1, and mcr–8 genes encoding
phosphoethanolamine transferase enzyme are also responsible for
plasmid-mediated Colistin resistance [35].
CONCLUSIONS
As a result, the most prevalent serotype among E. coli isolates
from chickens was serotype O78. The results also conrmed that
there is a variety of APEC strains in the poultry population. The PCR
assay used in this study, was able to differentiate APEC predominant
serotypes of O1, O2, O18 and O78 strains in accordance with serological
methods. Thus, implementation of this PCR assay benets for clinical
diagnostics, epidemiology studies, and disease control.
This APEC strains isolated in this study showed high prevalence
of resistance against Ampicillin, Cotrimoxazole, Doxycycline and
Ciprofloxacin isolated from the colibacillosis suspected broiler
chickens. However, none of the isolates were tested positive for
the mcr-1 and mcr–2 genes. The absence of the mcr-1 gene, which
is often responsible for plasmid-mediated transfer, is considered
promising for the Country. However, considering the present study
as a pilot study, it is important to conduct studies with a large number
of samples to investigate newly reported genes responsible for
Colistin resistance. Colistin is an important antibiotic, especially
as a last resort in both medical and veterinary elds. Therefore, it
is thought that studies in the eld of Veterinary Medicine, aimed at
limiting the use of Colistin and determining its place in the Global
problem of antibiotic resistance, are crucial. Another approach
should be improving surveillance and monitoring of multidrug-
resistant bacteria. This would involve the development of rapid
diagnostic tests to identify patients with Colistin-resistant infections,
as well as the implementation of surveillance programs to track
the spread of multidrug-resistant bacteria in healthcare settings
Molecular characterization of APEC isolated from broilers / Parin and Simsek ______________________________________________________
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and the environment. The development of new antibiotics and
alternative therapies are also important strategies for managing
Colistin resistant bacteria. Routine monitoring and screening of the
antibiotic resistance of avian pathogenic E. coli strains are crucial
for operating intervention programs to reduce risk of colibacillosis.
A holistic application is required for the prevention and the control
of avian colibacillosis in Western Cities and other regions of Turkey.
ACKNOWLEDGEMENT
This study was funded by Scientic Research Committee of Aydın
Adnan Menderes University with grant number of VTF-18033.
Conict of interest
The authors declare that they have no conict of interest.
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