DOI: https://doi.org/10.52973/rcfcv-e32159

Received: 23/05/2022 Accepted: 23/07/2022 Published: 28/08/2022

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Revista Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32159, 1 - 8

ABSTRACT

The study aims to identify phylogenetic groups and antibiotic

susceptibility of poultry Escherichia coli (APEC) isolates. E. coli was

phenotypically and biochemically characterized. Isolates from 8/30

(26.66%) liver, 7/30 (23.33%) heart, and 4/30 (13.33%) spleen of 37-42

days old vaccinated broiler chickens were assessed. Then the E. coli

isolates (19/90; 21.11%) were phylogrouped by quadruplex genotyping

based on the presence or absence of arpA, chuA, yjaA genes, and

TspE4.C2 DNA fragment.The majority of APEC strains belonged to

phylogenetic group C, followed by groups A, E, and F. Phylogroup C

was observed in the liver, phylogroup A in both liver and heart samples,

phylogroup E in the heart and spleen, and phylogroup F in the liver. The

highest antibiotic resistance was observed in Amoxicillin-Clavulanic

acid and Ampicillin (100%) predominantly in groups A and E according

to antibacterial susceptibility tests. Multiple antibiotic resistance

(MDR) for APEC strains was also found at 68.42% (13/19). Of the 19

isolates tested, only 13 (68%) were susceptible to high levels of

gentamicin. APEC strains belonging to phylogroups C, A, and E are

of epidemiological importance for broilers. It would be benecial to

investigate new phylogroups by performing more detailed genotypic

analyzes in APEC strains.

Key words: Broilers; molecular biology; Escherichia coli; phylogenetic;

susceptibility

RESUMEN

El estudio tuvo como objetivo identicar los grupos logenéticos y

la susceptibilidad a los antibióticos de los aislados de Escherichia

coli de aves de corral (APEC). E. coli se caracterizó fenotípica y

bioquímicamente a partir de hígado, 8/30 (26,66 %); corazón, 7/30

(23,33 %) y bazo, 4/30 (13,33 %) de pollos de engorde de 37-42 días

de edad vacunados. Luego, los aislados de E. coli (19/90; 21,11 %)

se loagruparon mediante genotipado cuádruple en función de la

presencia o ausencia de los genes arpA, chuA, yjaA y el fragmento de

ADN TspE4.C2. La mayoría de las cepas APEC pertenecían al grupo

logenético C, seguido de los grupos A, E y F. El logrupo C se observó

en el hígado, el logrupo A en las muestras de hígado y corazón,

el logrupo E en el corazón y el bazo y el logrupo F en el hígado.

La mayor resistencia antibiótica se observó en amoxicilina-ácido

clavulánico y ampicilina (100 %) predominantemente en los grupos

A y E según pruebas de susceptibilidad antibacteriana. También se

encontró resistencia múltiple a antibióticos (MDR) para las cepas

APEC en 68,42 % (13/19). De los 19 aislamientos probados, solo 13

(68 %) fueron susceptibles a niveles altos de gentamicina. Las cepas

APEC pertenecientes a los logrupos C, A y E son de importancia

epidemiológica para los pollos de engorde. Sería beneficioso

investigar nuevos logrupos realizando análisis genotípicos más

detallados en las cepas APEC.

Palabras clave: Pollos de engorde; biología molecular, Escherichia

coli; genética; microbiología

Phylogenetic characterization and determination of antibiotic

susceptibility of avian pathogenic Escherichia coli strains isolated from

broiler visceral organs

Caracterización logenética y determinación de la susceptibilidad a los antibióticos de cepas

patógenas de Escherichia coli aviar aisladas de órganos viscerales de pollos de engorde

Volkan Özavci

* , Haze Tuğba Yüksel-Dolgun

and Şükrü Kirkan

Dokuz Eylül University, Faculty of Veterinary Medicine, Department of Microbiology. Kiraz, Izmir, Turkey.

Aydin Adnan Menderes University, Faculty of Veterinary Medicine, Department of Microbiology. Işıklı, Aydin, Turkey.

*Email: volkan.ozavci@deu.edu.tr

Avian Pathogenic Escherichia Coli In Broilers / Özavci et al. _________________________________________________________________________

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INTRODUCTION

Escherichia coli is a pathogen implicated in intestinal and extraintestinal

infections [40]. Intestinal pathogens include enteropathogenic E.

coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli

(EHEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC),

and diffusely adherent E. coli (DAEC) [19]. Extra-intestinal pathogenic E.

coli (ExPEC) are an important group of pathogenic E. coli causing systemic

colibacillosis in poultry caused by avian pathogenic E. coli (APEC) and

responsible for economic losses for the world’s poultry industries

[11]. Some of the strains of E. coli found in the lower gastrointestinal

microbiota of poultry may spread viscerally and cause high morbidity

and mortality (20%) in chicken (Gallus gallus domesticus) ocks [9]. It

also causes loss of live weight (2%), decrease in feed eciency (2.7%)

and decrease in egg production (up to 20%) [20]. Avian pathogenic E.

coli (APEC) causes mostly systemic colibacillosis disease resulting in

signicant local or regional economic losses in the poultry industry [10,

30]. Wild ducks (Anas platyrhynchos), geese (Anser anser), and wild fowls

can be carriers of APEC and present a global threat to nutrition safety

and poultry welfare [27]. APEC is common in all ages of chickens (9.52

to 36.73%) [18]. Besides the air sacs, strains may also infect the organs

such as the spleen, peritoneum, pericardium, liver, yolk sac, pleura,

and oviduct [12]. Some virulence genes found in APEC belonging to the

phylogenetic group associated with extraintestinal pathogenic.

E. coli (ExPEC) have also been found in human ExPEC [13]. In addition,

APEC group D has been reported to mostly belong to a phylogenetic

group of ECOR. It has been reported that lesser virulent APECs and even

avirulent commensal E. coli can be transmitted to immunocompromised

avians [2]. Particularly, a positive relationship has been shown between

neonatal meningitis-causing E. coli (NMEC) and uropathogenic E. coli

(UPEC). It indicates that APEC strains can be potential zoonotic agents

[20, 25, 29]. APEC can share virulence factors with extraintestinal

pathogenic E. coli associated with humans, and that case raises

the possibility that APEC may play a role in some cases of human

disease [29, 30]. It has also been stated that APEC and extraintestinal

pathogenic E. coli (ExPEC) strains cause diseases in humans, and

improperly cooked chicken meat can cause foodborne infections [22].

The uptaking of APEC plasmids by common E. coli strains increases

virulence in infections (respiratory infections, septicaemia in poultry,

dying of chicken embryos) [39, 41].

In addition, APEC can also be isolated from rodents (Rattus). Also,

vertical transmission through contaminated eggs is also seen in infected

chickens [18]. The zoonotic potential of APEC or other pathotypes

isolated in chickens depends on the phenotypic characterization and

appraisal of common serotypes isolated from infected chickens [34].

Understanding the structure of E. coli showed that strains that refer

to different phylogroups may be related to the source of isolation

[6]. Phylogenetic studies are important for understanding the E. coli

population, strains, hosts, and the pathophysiology of the diseases

[6]. The phylogenetic analysis has reported that E. coli consists of A,

B1, B2, and D phylogenetic groups.

Also, it has been demonstrated that virulent extraintestinal E. coli

strains are clustered in the main group B2 and to a secondary extent

group D but conversely most of the commensal strains are associated

with group A or group B1 [16, 40]. Although group C is closely related to

group B1, it also includes strains of different genotypes. Group E was

dened as a new group comprising previously unclassied strains.

Group F was considered the very close group of B2 [5]. Antibiotics

(Tetracyclines, Sulfonamides, and Aminoglycosides) are used to control

colibacillus in chickens. However, increasing resistance to some groups

of antibiotics (-Lactams, Colistin, and Carbapenems) also limits the use

of antibiotics to control APEC infections in chickens [20].

The rapid spread of APEC to various visceral organs and the lead to

septicemia characterized by lesions in multiple organs require more

rational use of antimicrobials to reduce infection-related morbidity and

mortality [22]. In addition, the potential of APEC to cause extraintestinal

diseases in humans should also be considered [35]. Especially, currently,

studies on antimicrobial-resistant ExPEC and ESBL generating E. coli are

increasing [26]. This study aimed at the phylogenetic characterization

and determination of the antibiotic susceptibility proles of E. coli strains

isolated from internal organs (liver, heart, and spleen) of broiler chickens.

MATERIALS AND METHODS

Sample collection

Ninety swab samples were collected from the internal organs (liver,

heart and spleen) of 30 broiler chickens from different farms (average

capacity 5,000-20,000 chickens) located in the Aegian and Western

Black Sea Region in Turkey, between November 2021 and January

2022. Broiler chickens in these poultry houses are 39-45 days old

and weigh 2,000-2,500 grams (g). All chickens have been vaccinated

with commercial live vaccines such as Hipraclone® H120 (Spain),

CEVAC® IBD-L (France), Nobilis® Ma5+Clone 30 and Nobilis® IB 4-91

(Netherland). Collected swab samples were brought to Aydin Adnan

Menderes University, Veterinary Faculty, Microbiology Department

diagnostic laboratory in a cold chain with Stuart transport medium.

Identication of E. coli

Once in the laboratory, swabs samples were immediately plated onto

Columbia agar enriched with 5% sheep blood (Oxoid, Basingstoke,

United Kingdom (UK)), then Urinary Tract Infection (UTI) Brilliance

Clarity Chromogenic agar (Oxoid, Basingstoke, UK), and Brilliant

Green agar (Oxoid, Basingstoke, UK), and were incubated aerobically

at 37

C overnight. Identication was performed based on morphologic

properties, and biochemical analyses [urease (-), catalase (-), Voges

proskauer (-), indole (+), carbohydrate fermentation (+), methyl red

(+), citrate (-)], then conrmed using the BD Phoenix™ 50 automatic

identication appliance [Becton, Dickinson, and Company, Franklin

Lakes, New Jersey, United States (USA)]. All samples were freezer-

preserved in 50% glycerol brain heart infusion broth (Oxoid, Basingstoke,

UK) at -20°C (Bosch, Series 4, Germany) until subsequent analysis.

DNA extraction

Total Deoxyribonucleic acid (DNA) extraction of APEC isolates

which were identied with BD Phoenix™ 50 automatic identication

appliance was carried out by using the Thermo Scientic™ Genomic

DNA Purification Extraction Kit (Waltham, Massachusetts, USA),

according to manufacturer instructions. Extracted DNA was quantied

measured with the Nanodrop device (Maestrogen®, MN-913, Taiwan)

and results were recorded. The extracted DNA were freezer-kept in

cryotubes at -20°C (Bosch, Serie 4, Germany).

Polymerase chain reaction (PCR)

Phylogroups were determined by a PCR protocol developed and adapted

by Clermont et al. [6]. The primer sequences used for the PCR reactions

(HIMEDIA Prima Trio™ Thermal Cycler, India) are given in TABLE I.

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First, a quadruplex PCR reaction was performed on avian pathogenic

E. coli isolates, and then isolates in a particular phylogroup were

determined according to the results obtained. In addition, PCR analyzes

were performed on avian pathogenic E. coli isolates using group C-

and group E-specic primers. The DNA from ATCC 25922 was used

as a positive control for E. coli. Quadruplex and group C/E specic

PCR reaction was examined at a total volume of 25 microliters (µL)

including 2x Taq Mastermix (GenetBio®, South Korea) 12,5 µL; 10 pikomol

(pmol) each forward and reverse primer 0,4 µL; 50-100 nanograms (ng)

template DNA 2 µL and completed with nuclease-free water. Quadruplex

PCR condition; initial denaturation 95°C during 5 minutes (min) 1 cycle;

cyclic denaturation 95°C, 30 seconds (sec), annealing 59°C, 30 sec,

elongation 72°C, 30 sec for 30 cycles; nal elongation 72°C, 5 min one

cycle. Group C/E specic PCR condition; initial denaturation 95°C, 5

min 1 cycle; cyclic denaturation 95°C, 30 sec, annealing 59°C, 30 sec

for group C and 57°C, 30 sec for group E, elongation 72°C, 30 sec for

30 cycles; nal elongation 72°C, 5 min one cycle. PCR products were

examined on 1.5% agarose gel electrophoresis and visualized on the

imaging system (Vilber-Lourmat™-Innity VX2, France).

Phylogenetic grouping of APEC

Phylogroups of avian pathogenic E. coli isolates were determined

that depending on the results of the quadruplex screening and the

C, E clade PCRs [6].

Antibiotic susceptibility test of avian pathogenic E. coli isolates

Antimicrobial susceptibility testing (n=19) of APEC isolates was

performed by the Kirby-Bauer disk diffusion method on Mueller

Hinton agar (MHA) at a 0.5 McFarland concentration [3]. Ampicillin

[2 micrograms (2µg)] (Oxoid, Hampshire, UK), Ciprofloxacin

(5µg), Enrooxacin (5µg), Tetracycline (30µg), Gentamicin (10µg),

Erythromycin (10µg), Sulfamethoxazole-Trimethoprim (25µg) and

Amoxicillin-Clavulanic acid (30µg) discs were used for antibiogram

tests. Inhibition zone diameters for Enterobacteriaceae were measured

and evaluated according to clinical and laboratory standards institute

(CLSI) [8].

TABLE I

List of primers used for phylotyping of avian pathogenic E. coli isolates

PCR reaction Primer Target Primer sequences

PCR product

(bp)

Quadruplex

chuA.a1

chuA

5’-ATGGTACCGGACGAACCAAC-3’

288

chuA.2 5’-TGCCGCCAGTACCAAAGACA-3’

yjaA.1b

yjaA

5’-CAAACGTGAAGTGTCAGGAG-3’

211

yjaA.2b 5’-AATGCGTTCCTCAACCTGTG-3’

TspE4C2.1b

TspE4.C2

5’-CACTATTCGTAAGGTCATCC-3’

152

TspE4C2.2b 5’-AGTTTATCGCTGCGGGTCGC-3’

AceK.f

arpA

5’-AACGCTATTCGCCAGCTTGC-3’

400

ArpA1.r 5’-TCTCCCCATACCGTACGCTA-3’

Group E

ArpAgpE.f

arpA

5’-GATTCCATCTTGTCAAAATATGCC-3’

301

ArpAgpE.r 5’-GAAAAGAAAAAGAATTCCCAAGAG-3’

Group C

trpAgpC.1

trpA

5’-AGTTTTATGCCCAGTGCGAG-3’

219

trpAgpC.2 5’-TCTGCGCCGGTCACGCCC-3’

Reference (Clermont et al. [6])

RESULTS AND DISCUSSION

Phenotypic identication

From a total of 90 liver, spleen, and heart swab samples examined

in this study. Nineteen (21.1%) APECs (liver: 8; heart: 7; spleen: 4)

were identied, and they were conrmed with the BD Phoenix™ 50

automatic identication device (Becton, Dickinson, and Company,

Franklin Lakes, New Jersey, USA). Of the 19 APEC isolates, twelve

were from one organ, three were from liver and spleen organs, two

were from a heart organ, and two were from liver and spleen organs.

Different APEC strains isolated from the same sample were subjected

to phylotyping separately.

Phylogenetic grouping of APEC isolates

As a result of phylotyping of 19 (21.1%), APEC isolates were identied

by the quadruplex PCR method and 7 (36.8%) of them were typed as

a single phylogroup. Two stage phylogroup typing was performed for

the remaining 12 (63.2%) APEC isolates. Uniform phylogrouping was

carried out according to the results obtained by PCR tests for groups

E and C of these 12 APEC isolates. As a result, 7 (36.8%) of 19 APEC

isolates were APEC phylogroup C, 5 (26.3%) were APEC phylogroup A,

and 5 (26.3%) were APEC phylogroup E, and 2 (10.6%) were typed as

APEC phylogroup F and their agarose gel electrophoresis micrograph

were given in FIG 1. The distribution of phylogrouped 19 APEC isolates

were shown in TABLE II.

Considering the distribution of phylogenetic groups of 19/90 (21.11%)

APEC strains identied in this study, 5 APEC isolates were isolated to

phylogroup A from 2 livers, 2 hearts, and 1 spleen. Also, 7 APEC isolates

were isolated to phylogroup C from 4 livers, 2 hearts, 1 spleen, 5 APEC

isolates were isolated to phylogroup E from 3 hearts, 2 spleens, and 2

APEC isolates were isolated to phylogroup F from 2 livers. There was

not any E. coli strains isolated in the 71 (78.88%) samples.



arpAarpA (400 bp) / trpA (Group C)

arpAarpA (400 bp) / chuA (288 bp) / yjaA ( 211 bp) / arpA

arpA (400 bp) / arpAchuA (288 bp);

chuAE. coli standard strain (ATCC 25922)

Avian Pathogenic Escherichia Coli In Broilers / Özavci et al. _________________________________________________________________________

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TABLE II

Phylogroup distributions of APEC isolates according to quadruplex, group E, group C PCR results

Sample number

of APEC isolates

Organs

Sample

Quadruplex PCR

Pre-Phylogroup

Results

Group E Group C

Final Phylogroup

Results

arpA chuA yjaA  arpA trpA

1 Liver + - - - A - - A

3 Heart + - + - A / C - + C

6 Heart + - - - A - - A

10 Liver + - + - A / C - + C

12 Liver + - + - A / C - + C

13 Spleen + + + - E / clade I + - E

14L Liver + - - - A - - A

14S1 Spleen + - - - A - - A

14S2 Spleen + - + - A / C - + C

17 Heart + + - - D / E + - E

21 Heart + + - - D / E + - E

22H1 Heart + - - - A - - A

22H2 Heart + - + - A / C - + C

23L Liver + - + - A / C - + C

23S Spleen + + - - D / E + - E

25 Liver - + - - F - - F

27 Liver - + - - F - - F

28 Liver + - + - A / C - + C

29 Heart + + - - D / E + - E

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Antimicrobial susceptibility testing of E. coli isolates

The antibiogram tests were applied to (n=19) identified APEC

isolates. APEC strains isolated from liver (1), heart (6) and spleen

(1) were 100% resistant to all tested antibiotics. Strains isolated

from liver (8), heart (7) and spleen (4) were also 100% resistant to

Ampicillin and Amoxicllin-Clavulanic acid. In addition, following

these percentages, other resistances rates were found as 87.5% for

Ampicillin, Ciprooxacin, Enrooxacin, Erythromycin, Tetracycline,

Sulfamethoxazole-Trimethoprim, and Amoxicllin-Clavulanic acid in

the liver (4), heart (2), and spleen (1), 75% for Ampicillin, Ciprooxacin,

Enrooxacin, Erythromycin, Tetracycline, and Amoxicllin-Clavulanic

acid in the liver (1), and spleen (2), respectively. In contrast, antibiotic

susceptibility rates were identied as 75% for Gentamicin in the liver

(6), heart (3) and spleen (4), 12.5% both Ciprooxacin and Enrooxacin

in the liver (1), and 25% for Sulfamethoxazole-Trimethoprim in the

spleen (2) (TABLE III).

from heart samples showed resistance to all antibiotics. Multiple

antibiotic resistance (MDR) APEC strains were also found as 68.42%

(13/19). Of the 19 isolates tested, only 13 (68%) were susceptible to high

levels of Gentamicin. Multidrug resistance proles of APEC isolates

were shown in TABLE IV.

TABLE III

General results of antibiotic susceptibility tests of APEC isolates



Isolate

Name

 CIP ENR E GEN TE  

1. L1 R R R R S R R R

2. L10 R R R R S R I R

3. L12 R S S R S R R R

4. L14 R R R R R I R R

5. L23 R R R R S R R R

6. L25 R R R I S R R R

7. L27 R R R R S R R R

8. L28 R R R R R R R R

9. H3 R R R R S R R R

10. H6 R R R R R R R R

11. H17 R R R R R R R R

12. H21 R R R R R R R R

13. H22A R R R R R R R R

14. H22B R R R R S R R R

15. H29 R R R R S R R R

16. S13 R R R R S R R R

17. S14A R R R R S R S R

18. S14B R R R R S R R R

19. S23 R R R R S R S R

L, liver; H, heart; S, spleen; R, resistance; I, intermediate; S, susceptible;

(AMP) Ampicillin 2 µg; (CIP) Ciprooxacin 5 µg; (ENR) Enrooxacin 5 µg;

(E) Erythromycin 10 µg; (GEN) Gentamicin 10 µg; (TE) Tetracycline 30 µg;

(TMP-SMX) Sulfamethoxazole-Trimethoprim 25 µg; (AMC) Amoxicillin-

Clavulanic acid 30 µg

It was determined that APEC strains were 100% resistant to Amoxicillin-

Clavulanic acid and Ampicillin, 94.7% to Ciprooxacin, Erythromycin,

Enrooxacin, Tetracycline, and 84.22% to Sulfamethaxazole-Trimethprim,

respectively. Generally, APEC strains showed 84% or more resistance to

7 antibiotics used in the research. Especially isolates (n=8/19) obtained

TABLE IV





Antibiotics

Antibiotic



Name of

Isolates



Isolates

Origin of

Isolates

AMP, CIP, ENR, E, GEN,

TE, TMP-SXM, AMC

L28, H6, H17,

H21, H22A

Liver(1),

Heart(4)

AMP, CIP, ENR, E, TE,

TMP-SXM, AMC

H3, H22B, H2,

S13, S14B

Heart (3),

Spleen(2)

AMP, CIP, ENR, E, TE,

TMP-SXM, AMC

L1, L23, L27 3 Liver

AMP, CIP, ENR, E,

GEN, TMP-SXM, AMC

L14 1 Liver

AMP, CIP, ENR,

E, TE, AMC

L10, S14A, S2 3

Liver(1),

Spleen(2)

AMP, CIP, ENR, TE,

TMP-SXM, AMC

L25 1 Liver

AMP, E, TE,

TMP-SXM, AMC

L12 1 Liver

L, liver; H, heart; S, spleen; R, resistance; I, intermediate; S, susceptible;

(AMP) Ampicillin 2 µg; (CIP) Ciprooxacin 5 µg; (ENR) Enrooxacin 5 µg;

(E) Erythromycin 10 µg; (GEN) Gentamicin 10 µg; (TE) Tetracycline 30 µg;

(TMP-SMX) Sulfamethoxazole-Trimethoprim 25 µg; (AMC) Amoxicillin-

Clavulanic acid 30 µg

Avian pathogenic E. coli causes major localized or systemic losses

in the poultry industry characterized by colisepticemia. It also

concerns public health as potential zoonotic agents [29, 30]. E. coli is

at least eight phylogenetic groups and is divided into three clusters:

phylogroups B2, G, and F, phylogroups A, B1, C, and E, and phylogroup

D (phylogroup D, the closest group to E. coli origin). Virulence genes

are mostly associated with phylogroups D and B2 [5, 27].

APEC an opportunistic pathogen can cause secondary infections in

visceral organs such as Newcastle disease, and Mycoplasmosis [15].

Phylogroup C is considered a different strain group closely related to

phylogroup B1. It is stated that group F consists of strains very close

to the B2 phylogroup. The inclusion of arpA has it possible to identify

the misidentied strain D phylogroup (chuA+, yjaA-, TspE4.C2-), which

should have previously belonged to F.

These genes out of the strains, due to they are present in all E. coli

strains rather than the strains which belong to B2 and F phylogroups,

should be differentiated [7]. In this study, extended quadruple PCR

was preferred as the phylogroup assignment method because it

provides advantages in detecting strains belonging to C, E, F, and

clade I phylogroups, although a small part of E. coli strains cannot

be included in a phylogroup and has disadvantages such as variable

gene content [6]. Commensal E. coli belongs to phylogenetic group

A. Groups D, most closely related to E. coli, consist of several

evolutionary lineages considered “virulent clones” [17].

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APEC strains were isolated and identied from liver, heart, and

spleen organ swaps collected from 2,000-2,500 g of 39-45 days old

broiler chickens. In this study, E. coli isolates (19/90; 21.11%) obtained

from chicken viscera showed similarity to previously reported

studies [34, 37]. Logue et al. [24] has been reported the distribution

of phylogenetic groups of 452 APEC strains isolated from poultry

according to the quadruplex PCR method (Clermont et al. [5]).

The rates of the phylogroups was A (10.17%), C (27.65%), D (5.08%), E

(3.31%), and F (19.26%), respectively. Ungrouped strains were reported

as 0.22%. In other studies, the D phylogenetic group was specied as

commonly found strains [4, 21]. As a result of this study, the A, C, and

E groups were found to be higher than the results they obtained. The

number of strains not included in any phylogroup was determined at a

higher rate (APEC; 78.88%). D and B2 are Groups of E. coli responsible

for extraintestinal diseases.

Phylogenetically, Group E is closely related to Group D (including

O157: H7), and also Group F is closely related to B2 [38]. In this study,

group F was found to be 10.52% among APEC strains. Subsequently,

according to the study by Coura et al. [9], higher results were observed

for Groups A (2.66%), F (3.33%), and E (12.00%), but similar results

were observed for Groups D (0.00%).

E. coli isolates in phylogroups A and D were reported to have spread

from breeders to broilers, and strains of phylogroup B2 and D were

accepted as extraintestinal pathogenic E. coli [2, 28, 33]. In this study,

phylotyping of strain A was made, but no phylotyping of extraintestinal

Group D was observed. However, the closely related Group E was

isolated. Phylogroups D and A are the dominant phylogroup in APEC

types [5, 6, 11, 41]. The obtained results support the knowledge that A

phylogroup is the most common strains obtained from broiler chickens

[9]. However, unlike Coura et al. [9] the obtained ndings highlight that

phylogroups C, E, and F can also be isolated from broilers.

APEC isolates were tested with 8 antimicrobial agents to examine

antibiotic susceptibility. Resistance was observed to Ampicillin and

Amoxicillin-Clavulanic acid (19/19; 100%), followed by Ciprooxacin,

Enrooxacin, Erythromycin, and Tetracycline (18/19; 94,7%), and nally

to Sulfamethoxazole-Trimethoprim (16/19; 84,21%) and Gentamicin

(6/19; 31,58%). In addition, susceptibility was observed for Gentamicin

(13/19; 68.42%), Sulfamethoxazole-Trimethoprim (2/19; 10.52%),

Ciprooxacin, Enrooxacin, and Erythromycin (1/19; 5.26%) in isolates.

Various studies have been conducted on resistance and

susceptibility. It has been reported that APEC isolates were resistant to

Sulfamethoxazole-Trimethoprim (95.5%), Amoxicillin (93.3%), Ampicillin

(89.6%), Amoxicillin-Clavulanate (79%), Gentamicin (68.8%), Ciprooxacin

(47.9%), and Tetracycline (45%) [14, 36]. The obtained results conrm the

resistance but draw attention to the high rates of resistance, especially

to Amoxicillin-Clavulanate, Ampicillin, Tetracycline, and Ciprooxacin.

It has been reported that discontinuing the non-therapeutic use of

antibiotics (such as Tetracyclines) prescribed to promote growth in

poultry led to a signicant reduction in resistant bacteria, which can

be seen especially in poultry [23]. Therefore, it is important not to use

unnecessary antibiotics to prevent the development of resistance.

The high APEC resistance to Tetracycline has been reported [31,

32]. The present ndings also conrm this evaluation. In a study,

the highest percentage of sensitivity for Enrofloxacin (53.85%)

and Gentamicin (46.15%) has been reported [1], but in this study,

the sensitivity was determined only at the Gentamicin level, and,

Enrooxacin was not found to be very effective. The high levels of

resistance observed for the antibiotic classes in this study suggest

that they are widely used in the avian industry. Besides, it was pointed

out that the origin of APEC strains may be poultry material and may

be important in infecting other animals by being present in the

environment.

CONCLUSIONS

This study demonstrated that APEC strains could be isolated from

edible organs. Also, strains may spread from poultry residuals to the

environment and may transmit to other animals in this way. Escherichia

coli poses a serious threat to human health and food safety.

In the present study, phylogroup C was the most common group.

Phylogroups A and E were generally isolated from the heart and spleen,

while phylogroups C and F were detected from liver and heart samples.

For this reason, ensuring hygiene in the poultry houses is important

both in terms of poultry health and providing safe food to people.

The indiscriminate use of antibiotics in animal production may

contribute to the resistant strains of APEC strains. Because of its

zoonotic importance, routine laboratory research targeting APEC

virulence genes for the early detection of avian colibacillosis may be

benecial in preventing unnecessary antibiotic use and resistance. Also,

It would be benecial to investigate new phylogroups by performing

more detailed genotypic analyzes in APEC strains.

ACKNOWLEDGEMENTS

This study has not received any funding support. We would like

to present our gratitude to all veterinary colleagues and valuable

producers for their help in obtaining samples collected from poultry

carcasses. The authors declare no conict of interest for this study.

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