Virulence and Antimicrobial Resistance of Avian Pathogenic Escherichia coli (APEC) Isolates from Poultry in Brazil

Abstract

Colibacillosis is a chicken disease caused by avian pathogenic Escherichia coli (APEC). Pathogenicity in birds is determined by the occurrence of bacterial genes encoding virulence factors in APEC strains. Furthermore, APEC and other bacterial infections in commercial poultry farms have been treated with intensive use of antimicrobials for decades. Currently, many APEC strains are no longer susceptible to frequently used antibiotics due to increasing antimicrobial resistance (AMR) associated with the acquisition and mutation of other specific bacterial genes. The present study aimed to isolate and detect APEC isolates in broiler farms from different poultry-producing regions of Brazil and to determine their AMR profile. A total of 126 E. coli isolates were obtained from necropsied chickens with colibacillosis. All of these E. coli isolates were analyzed with one species-specific qPCR (targeting uspA gene) and five virulence factors genes qPCRs (targeting iroN, hlyF, iutA, iss, and ompT). AMR was determined by disk diffusion method using ten drugs frequently used to treat colibacillosis in Brazilian poultry farms. The results demonstrated that 109 (86.5%) isolates were classified as APEC. AMR was commonly observed in APEC and AFEC isolates, highlighting resistance for amoxicillin (85; 67.4%) and ceftiofur (72; 57.1%). A total of 41 (32.5%) E. coli isolates presented a multidrug resistance (MDR) profile. These results can contribute to implementing more effective colibacillosis prevention and control programs on Brazilian poultry farms.

Full text

Academic Editor: Alessandra
Piccirillo
Received: 29 November 2024
Revised: 11 February 2025
Accepted: 21 February 2025
Published: 24 February 2025
Citation: Lúcio, C.J.; Hansen, P.H.C.;
Griebeler, J.; Kipper, D.; Lunge, V.R.
Virulence and Antimicrobial
Resistance of Avian Pathogenic
Escherichia coli (APEC) Isolates from
Poultry in Brazil. Poultry 2025,4, 10.
https://doi.org/10.3390/
poultry4010010
Copyright: © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/
licenses/by/4.0/).
Article
Virulence and Antimicrobial Resistance of Avian Pathogenic
Escherichia coli (APEC) Isolates from Poultry in Brazil
Caio Júnior Lúcio 1, Paulo Henrique Caminha Hansen 1, Josiane Griebeler 1, Diéssy Kipper 2
and Vagner Ricardo Lunge 1,2, *
1Molecular Diagnostic Laboratory, Lutheran University of Brazil (ULBRA), Canoas 92425-900, RS, Brazil;
caio.jlucio@gmail.com (C.J.L.); paulohch2@gmail.com (P.H.C.H.); jogriebeler@gmail.com (J.G.)
2Simbios Biotecnologia, Cachoeirinha 94940-030, RS, Brazil; diessykipper@hotmail.com
*Correspondence: lunge@ulbra.br
Abstract: Colibacillosis is a chicken disease caused by avian pathogenic Escherichia coli
(APEC). Pathogenicity in birds is determined by the occurrence of bacterial genes encoding
virulence factors in APEC strains. Furthermore, APEC and other bacterial infections
in commercial poultry farms have been treated with intensive use of antimicrobials for
decades. Currently, many APEC strains are no longer susceptible to frequently used
antibiotics due to increasing antimicrobial resistance (AMR) associated with the acquisition
and mutation of other specific bacterial genes. The present study aimed to isolate and detect
APEC isolates in broiler farms from different poultry-producing regions of Brazil and to
determine their AMR profile. A total of 126 E. coli isolates were obtained from necropsied
chickens with colibacillosis. All of these E. coli isolates were analyzed with one species-
specific qPCR (targeting uspA gene) and five virulence factors genes qPCRs (targeting iroN,
hlyF,iutA,iss, and ompT). AMR was determined by disk diffusion method using ten drugs
frequently used to treat colibacillosis in Brazilian poultry farms. The results demonstrated
that 109 (86.5%) isolates were classified as APEC. AMR was commonly observed in APEC
and AFEC isolates, highlighting resistance for amoxicillin (85; 67.4%) and ceftiofur (72;
57.1%). A total of 41 (32.5%) E. coli isolates presented a multidrug resistance (MDR) profile.
These results can contribute to implementing more effective colibacillosis prevention and
control programs on Brazilian poultry farms.
Keywords: APEC; E. coli; poultry; Brazil; MDR; AMR; colibacillosis
1. Introduction
Escherichia coli is generally a commensal microorganism in homothermic animals,
being present in the intestinal microbiota of mammals and birds [
1
]. In chickens, enteric
colonization by E. coli occurs soon after birth, an important process to help protect the
intestine against pathogenic bacteria [
2
]. However, there are also pathogenic strains of E.
coli that infect chickens and cause clinical manifestations, such as pericarditis, airsacculitis,
perihepatitis, peritonitis, synovitis, salpingitis, and osteomyelitis. Whenever these diseases
are caused by E. coli strains, with identification of this bacterial species, they are called
colibacillosis [3,4].
Colibacillosis occurs only in birds infected by avian pathogenic E. coli (APEC) strains.
On the contrary, non-pathogenic strains are known as AFEC (avian fecal E. coli) and they
are present in healthy chicken enteric tracts [
3
,
5
]. Differentiation between APEC and AFEC
strains is necessary for routine laboratory diagnostic testing, and it is currently performed
by detection of some virulence genes usually hosted in E. coli plasmids and chromosomal
Poultry 2025,4, 10 https://doi.org/10.3390/poultry4010010

Poultry 2025,4, 10 2 of 11
DNA. In a pivotal study, 46 potential virulence genes were studied in E. coli isolates from
poultry farms. APEC strains usually possess at least five main virulence genes: iroN,hlyF,
iutA,iss, and ompT (salmochelin siderophore receptor gene, putative avian hemolysin,
aerobactin siderophore receptor gene, episomal enhanced serum survival gene, and outer
membrane protease gene, respectively) [
5
]. Based on this study, PCR (Polymerase Chain
Reaction) assays were developed to detect these five genes for a diagnostic procedure to
identify APEC isolates. This method has been previously used in poultry farms worldwide,
including Brazil [3,6–8].
Antimicrobials have been used as therapeutic agents to treat bacterial infections caused
by pathogenic bacteria (such as Salmonella,Clostridium, APEC, etc.) responsible for chicken
diseases with high mortality rates and significant production losses in poultry farms
worldwide. Ampicillin, ciprofloxacin, streptomycin, sulfamethoxazole, and tetracycline
are some of the most used antimicrobials in poultry farming [
9
]. As a consequence, the
frequency of E. coli isolates resistant to different antimicrobials has increased in the last
decades. In Brazil, resistance to
β
-lactams, tetracyclines, quinolones, and sulfonamides has
been reported in E. coli isolates from poultry farms [8,10–12].
This study aimed to investigate the frequency of APEC isolates and to evaluate E. coli
resistance to the most common antimicrobial used in broiler farms from important Brazilian
poultry-producing regions in the last years.
2. Materials and Methods
2.1. Sampling
The E. coli isolates were obtained by convenience sampling in a routine laboratory of
an agro-industrial company in Brazil between March 2022 and March 2023. All them were
obtained after bacteriological analysis of tissue pools from necropsied chickens (5 to 10 per
flock) presenting clinical signs of colibacillosis (pericarditis, airsacculitis, perihepatitis). A total
of 126 (14.5%) E. coli isolates were obtained after the analysis of tissues from 870 necropsied
chickens in the laboratory. These chicken samples were from 126 different broiler flocks
located on commercial poultry farms from three important poultry-producing states in Brazil:
Minas Gerais (n= 31), Paraná (n= 55), and Rio Grande do Sul (n= 40) (Figure 1).
Poultry 2025, 4, x FOR PEER REVIEW 3 of 12
Figure 1. Map of Brazil demonstrating the different states where the 126 E. coli isolates were ob-
tained. Pie graphs present the frequencies of APEC and AFEC isolates in each state.
2.2. Bacteriological Procedures
E. coli isolates were obtained from the necropsied organs (liver and heart) macerated
and directly streaked on eosin methylene blue agar (EMB; BD DifcoTM; Franklin Lakes,
NJ, USA). Plates were incubated at 36 °C for 16 to 24 h. E. coli characteristic colonies were
transferred to a tube with brain heart infusion medium (BHI Broth; Merck™, Darmstadt,
Germany) plus 10% glycerol and stored in a freezer. To recover the isolates for this study,
100 µL of the stored culture was transferred to a tryptone soy broth (TSB; BD DifcoTM;
Franklin Lakes, NJ, USA) and incubated at 36 °C for 24 h. Subsequently, each E. coli strain
was seeded on nutrient agar (BD DifcoTM; Franklin Lakes, NJ, USA) and incubated at 36
°C for 18 to 24 h. Typical E. coli colonies were used in the next experiments.
2.3. DNA Extraction and E. coli Identification
DNA was extracted from all E. coli isolates as previously described [13]. Briefly, a
pick of each typical E. coli colony was suspended in a microtube containing 100 µL of ul-
trapure water (Nova Biotecnologia, Cotía, Brazil) and heated to a temperature of 100 °C
for 10 min, after quickly cooling in a cold block to −20 °C for 5 min and centrifugation at
12,000 rpm for 2 min.
As previously described, E. coli isolate DNA was first analyzed with a spe-
cies-specific qPCR targeting uspA (universal stress protein A) [14]. Briefly, 4 µL of the
extracted DNA was transferred to microplates containing 5 µL of the commercial su-
permix SsoFastTM EvaGreen
®
(Bio-Rad Laboratories, Hercules, CA, USA) and 2.5 nM of
the two specific primers (For 5’-CGC TGC CAA TCA GTT AAC ACC-3′; Rev 5’-CGT
CAA TCC GCC TTG CTT AC-3’). qPCR assays were carried out in a CFX 96TM ther-
mocycler (Bio-Rad Laboratories, Hercules, CA, USA) with the following amplification
conditions: 1 cycle of 95 °C for 3 min and 34 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72
°C for 15 s. Ct (Cycle threshold) and Tm (Temperature melting) were analyzed with
graphs generated by the Bio-Rad MaestroTM software—version 5.2.008.0222. Negative
(water) and positive (DNA of E. coli ATCC
®
25922TM strain) were included in all runs.
2.4. Virulence Genes Detection by qPCR
Five main E. coli virulence genes (iroN, hlyF, iutA, iss, and ompT) were detected by
qPCR (Real-Time PCR) as previously described [5]. All PCRs were carried out in a total
Figure 1. Map of Brazil demonstrating the different states where the 126 E. coli isolates were obtained.
Pie graphs present the frequencies of APEC and AFEC isolates in each state.

Poultry 2025,4, 10 3 of 11
2.2. Bacteriological Procedures
E. coli isolates were obtained from the necropsied organs (liver and heart) macerated
and directly streaked on eosin methylene blue agar (EMB; BD DifcoTM; Franklin Lakes,
NJ, USA). Plates were incubated at 36 ◦C for 16 to 24 h. E. coli characteristic colonies were
transferred to a tube with brain heart infusion medium (BHI Broth; Merck™, Darmstadt,
Germany) plus 10% glycerol and stored in a freezer. To recover the isolates for this study,
100
µ
L of the stored culture was transferred to a tryptone soy broth (TSB; BD DifcoTM;
Franklin Lakes, NJ, USA) and incubated at 36
◦
C for 24 h. Subsequently, each E. coli strain
was seeded on nutrient agar (BD DifcoTM; Franklin Lakes, NJ, USA) and incubated at
36 ◦C for 18 to 24 h. Typical E. coli colonies were used in the next experiments.
2.3. DNA Extraction and E. coli Identification
DNA was extracted from all E. coli isolates as previously described [
13
]. Briefly, a pick
of each typical E. coli colony was suspended in a microtube containing 100
µ
L of ultrapure
water (Nova Biotecnologia, Cotía, Brazil) and heated to a temperature of 100
◦
C for 10 min,
after quickly cooling in a cold block to
−
20
◦
C for 5 min and centrifugation at 12,000 rpm
for 2 min.
As previously described, E. coli isolate DNA was first analyzed with a species-specific
qPCR targeting uspA (universal stress protein A) [
14
]. Briefly, 4
µ
L of the extracted DNA
was transferred to microplates containing 5
µ
L of the commercial supermix SsoFastTM
EvaGreen
®
(Bio-Rad Laboratories, Hercules, CA, USA) and 2.5 nM of the two specific
primers (For 5
′
-CGC TGC CAA TCA GTT AAC ACC-3
′
; Rev 5
′
-CGT CAA TCC GCC
TTG CTT AC-3
′
). qPCR assays were carried out in a CFX 96TM thermocycler (Bio-Rad
Laboratories, Hercules, CA, USA) with the following amplification conditions: 1 cycle of
95 ◦C
for 3 min and 34 cycles of 95
◦
C for 10 s, 60
◦
C for 10 s, and 72
◦
C for 15 s. Ct (Cycle
threshold) and Tm (Temperature melting) were analyzed with graphs generated by the
Bio-Rad MaestroTM software—version 5.2.008.0222. Negative (water) and positive (DNA
of E. coli ATCC®25922TM strain) were included in all runs.
2.4. Virulence Genes Detection by qPCR
Five main E. coli virulence genes (iroN,hlyF,iutA,iss, and ompT) were detected by
qPCR (Real-Time PCR) as previously described [
5
]. All PCRs were carried out in a total
volume of 10
µ
L per reaction with 4
µ
L of the E. coli DNA previously extracted, 5
µ
L of the
commercial supermix SsoFastTM EvaGreen
®
(Bio-Rad Laboratories, Hercules, CA, USA),
and 1
µ
L of the primer solution (2.5 nM). All PCRs were also carried out in a CFX 96TM
thermocycler (Bio-Rad Laboratories, Hercules, CA, USA) with the following amplification
conditions: 1 cycle of 95
◦
C for 3 min, 40 cycles of 95
◦
C for 10 s, and 57
◦
C for 30 s.
All results were reported as Ct and Tm graphs generated using the Bio-Rad MaestroTM
software—version 5.2.008.0222. DNA from one APEC isolate positive for the five virulence
genes was used as a positive control in all assays.
E. coli isolates were classified into APEC according to the presence of three or more
virulence genes. In addition, a comparative analysis considering three, four, and five genes,
as APEC, was also carried out according to other studies [8,10,15,16].
2.5. Antimicrobial Susceptibility Testing
The antimicrobial susceptibility testing of the isolates was carried out using the Mueller–
Hinton agar disk diffusion method (AMH; OxoidTM Thermo Fisher Scientific Inc., Waltham,
MA, USA) as previously described [
17
]. After turbidity of 0.5 on a McFarland scale, each
E. coli isolate sample was uniformly spread using a sterile cotton swab. After, antimicrobial
discs were placed and lightly pressed on the plate surface. All plates were incubated at
36 ◦C

Poultry 2025,4, 10 4 of 11
for
16 to
18 h. The following antimicrobials from the manufacturer Oxoid (Thermo Fisher
Scientific Inc., Waltham, MA, USA) were used: Amoxicillin (AMX), [10
µ
g]; Ceftiofur (CEF),
[30
µ
g]; Ciprofloxacin (CIP), [5
µ
g]; Doxycycline (DO), [30
µ
g]; Enrofloxacin (ENR), [5
µ
g];
Florfenicol (FFC), [30
µ
g]; Gentamicin (GM), [10
µ
g]; Neomycin (NEO), [10
µ
g]; Norfloxacin
(NX), [10
µ
g]; and Tetracycline (TE), [30
µ
g]. All experimental procedures were also carried
out with the E. coli ATCC®25922TM reference strain as a control.
The results were evaluated by measuring the diameter of the inhibition zones using
a millimeter ruler. The interpretation of the results of most antimicrobials followed the
criteria defined by the Clinical and Laboratory Standards Institute—CLSI [
17
,
18
]. In the
analysis of the results for neomycin and florfenicol, criteria from other two publications
were also used [
19
,
20
]. The isolates were considered MDR (multidrug resistance) if they
were resistant to at least one agent in three or more chemical classes of antimicrobials [
21
].
2.6. Statistical Analysis
Fisher’s exact test was used to compare the frequencies of virulence factors between
isolates classified as APEC and AFEC. A p-value of <0.05 was considered statistically
significant. SPSS
®
Statistics version 25 software was used to test the null hypothesis of
equality between gene frequencies (IBM Corporation, Armonk, NY, USA).
3. Results
3.1. APEC and AFEC Isolate Detection
All 126 isolates were identified as E. coli according to uspA gene positive results. In the
differentiation of APEC and AFEC, 115 (91.3%) isolates were classified as APEC since they
presented positive results for three or more virulence genes analyzed. The total number
and respective frequency of APEC isolates varied according to the state of the origin of
the samples: 30 (96.8%) in Minas Gerais, 34 (85%) in Rio Grande do Sul, and 45 (81.8%) in
Paraná (Figure 1).
In an additional separate analysis of the virulence gene frequencies, qPCR results
showed that the hlyF and ompT genes were the most frequent ones (89.7%), followed by
iroN (84.9%), iss (84.1%), and iutA (80.2%) (Table 1).
Table 1. Virulence genes detected in the E. coli isolates according to the origin of the samples (Minas
Gerais—MG, Paraná—PR, and Rio Grande do Sul—RS).
Gene MG (n = 31) PR (n = 55) RS (n = 40) Total (n= 126)
n(%) n(%) n(%) n(%)
iroN 30 (96.8) 44 (80) 33 (82.5) 107 (84.9)
hlyF 29 (93.5) 49 (89.1) 35 (87.5) 113 (89.7)
iutA 29 (93.5) 44 (80) 28 (70) 101 (80.2)
iss 30 (96.8) 45 (81,8) 31 (77.5) 106 (84.1)
ompT 30 (96.8) 48 (87.7) 35 (87.5) 113 (89.7)
All five virulence genes were detected in 76.2% of the isolates (96/126), while none
of these same genes were detected in 7.1% of the isolates (9/126). In a comparison of the
Brazilian states, Minas Gerais presented the highest positive isolates number for all five
genes (30/31; 96.8%). A heat map was created to demonstrate the presence/absence of the
five genes in the isolates from the different farms (Figure 2).

Poultry 2025,4, 10 5 of 11
Poultry 2025, 4, x FOR PEER REVIEW 5 of 12
Table 1. Virulence genes detected in the E. coli isolates according to the origin of the samples (Mi-
nas Gerais—MG, Paraná—PR, and Rio Grande do Sul—RS).
Gene MG (n = 31) PR (n = 55) RS (n = 40) Total (n = 126)
n (%) n (%) n (%) n (%)
iroN 30 (96.8) 44 (80) 33 (82.5) 107 (84.9)
hlyF 29 (93.5) 49 (89.1) 35 (87.5) 113 (89.7)
iut
A
29 (93.5) 44 (80) 28 (70) 101 (80.2)
iss 30 (96.8) 45 (81,8) 31 (77.5) 106 (84.1)
ompT 30 (96.8) 48 (87.7) 35 (87.5) 113 (89.7)
All five virulence genes were detected in 76.2% of the isolates (96/126), while none of
these same genes were detected in 7.1% of the isolates (9/126). In a comparison of the
Brazilian states, Minas Gerais presented the highest positive isolates number for all five
genes (30/31; 96.8%). A heat map was created to demonstrate the presence/absence of the
five genes in the isolates from the different farms (Figure 2).
In addition, more rigorous criteria to determine APEC were used to evaluate all
isolates (occurrence of four or five virulence genes). A total of 103 (81.7%) and 96 (76.2%)
E. coli isolates were considered APEC with four and five genes, respectively. In Paraná,
80% (44/55) of the isolates presented four or five, and 70.9% (39/55) had all five genes. In
Rio Grande do Sul, 75% (30/40) of the isolates presented four or five, and 70% (28/40) had
all five genes. In Minas Gerais, 93.5% (29/31) of the isolates had four and five genes (Table
2).
Figure 2. Relationship between states, APEC, AFEC and virulence genes of the 126 E. coli isolates.
The first column indicates the isolate origin state. The second column shows the classification into
APEC (blue) or AFEC (green). Columns 3 to 7 classify the strain according to the presence (black)
and absence (white) of the five virulence genes iroN, hlyF, iutA, iss, and ompT.
Table 2. Virulence gene frequency in APEC and AFEC isolates according to the presence of four
and five genes.
Gene APEC 2 n = 103
n (%)
AFEC n = 23
n (%) p-Value 1 APEC 3 n = 96
n (%)
AFEC n = 30
n (%) p-Value 1
iroN 102 (99.0) 5 (21.7) <0.05 96 (100) 11 (36.7) <0.05
hlyF 102 (99.0) 11 (47.8) <0.05 96 (100) 17 (56.7) <0.05
iutA 98 (95.1) 3 (13.0) <0.05 96 (100) 5 (16.7) <0.05
iss 103 (100) 3 (13.0) <0.05 96 (100) 10 (33.3) <0.05
ompT 103 (100) 10 (43.5) <0.05 96 (100) 17 (56.7) <0.05
1 The result is significant at p ≤ 0.05. 2 Minimum parameter of four genes. 3 Minimum parameter of
five genes.
Figure 2. Relationship between states, APEC, AFEC and virulence genes of the 126 E. coli isolates.
The first column indicates the isolate origin state. The second column shows the classification into
APEC (blue) or AFEC (green). Columns 3 to 7 classify the strain according to the presence (black)
and absence (white) of the five virulence genes iroN,hlyF,iutA,iss, and ompT.
In addition, more rigorous criteria to determine APEC were used to evaluate all
isolates (occurrence of four or five virulence genes). A total of 103 (81.7%) and 96 (76.2%) E.
coli isolates were considered APEC with four and five genes, respectively. In Paraná, 80%
(44/55) of the isolates presented four or five, and 70.9% (39/55) had all five genes. In Rio
Grande do Sul, 75% (30/40) of the isolates presented four or five, and 70% (28/40) had all
five genes. In Minas Gerais, 93.5% (29/31) of the isolates had four and five genes (Table 2).
Table 2. Virulence gene frequency in APEC and AFEC isolates according to the presence of four and
five genes.
Gene APEC 2n = 103
n(%)
AFEC n = 23
n(%) p-Value 1
APEC
3
n = 96
n(%)
AFEC n = 30
n(%) p-Value 1
iroN 102 (99.0) 5 (21.7) <0.05 96 (100) 11 (36.7) <0.05
hlyF 102 (99.0) 11 (47.8) <0.05 96 (100) 17 (56.7) <0.05
iutA 98 (95.1) 3 (13.0) <0.05 96 (100) 5 (16.7) <0.05
iss 103 (100) 3 (13.0) <0.05 96 (100) 10 (33.3) <0.05
ompT 103 (100) 10 (43.5) <0.05 96 (100) 17 (56.7) <0.05
1The result is significant at p≤0.05. 2Minimum parameter of four genes. 3Minimum parameter of five genes.
3.2. Antimicrobial Susceptibility
The antimicrobial susceptibility profile of all E. coli isolates demonstrated that 112
(88.9%) were resistant to at least one drug, 99 (78.6%) to at least two drugs, 74 (58.7%) to at
least three drugs, 58 (46%) to at least four drugs, 41 (32.5%) to at least five drugs, 19 (15.1%)
to at least six drugs, 10 (7.9%) to at least seven drugs, 5 (4%) to at least eight drugs, and 1
(0.8%) to at least nine drugs (Figure 3).
APEC isolates presented high frequency of resistance to amoxicillin (72/109; 66.1%),
followed by ceftiofur (62/109; 56.9%), norfloxacin (52/109; 47.7%), tetracycline (37/109;
33.9%), ciprofloxacin (34/109; 31.2%), florfenicol (28/109; 25.7%), doxycycline (26/109;
23.9%), enrofloxacin (25/109; 22.9%), neomycin (16/109; 14.7%), and gentamicin (3/109;
2.8%). In addition, there was also intermediate resistance for six antimicrobials: enrofloxacin
(46/109; 42.2%), ciprofloxacin (36/109; 33%), doxycycline (9/109; 8.2%), gentamicin (7/109;
6.4%), ceftiofur (5/109; 4.6%), and amoxicillin (2/109; 1.8%). A total of 32 (29.4%) APEC
isolates presented MDR profiles; 40.6% (13/32) were from Paraná, 34.4% (11/32) were
from Rio Grande do Sul, and 25% (8/32) were from Minas Gerais. The most frequent
MDR patterns observed in APEC were
β
-lactams + fluoroquinolones + tetracyclines and
β-lactams + fluoroquinolones + aminoglycosides + tetracyclines.

Poultry 2025,4, 10 6 of 11
Poultry 2025, 4, x FOR PEER REVIEW 6 of 12
3.2. Antimicrobial Susceptibility
The antimicrobial susceptibility profile of all E. coli isolates demonstrated that 112
(88.9%) were resistant to at least one drug, 99 (78.6%) to at least two drugs, 74 (58.7%) to
at least three drugs, 58 (46%) to at least four drugs, 41 (32.5%) to at least five drugs, 19
(15.1%) to at least six drugs, 10 (7.9%) to at least seven drugs, 5 (4%) to at least eight
drugs, and 1 (0.8%) to at least nine drugs (Figure 3).
Figure 3. Multiple and individual profiles of antimicrobial susceptibility testing in 126 E. coli iso-
lates. The first column indicates the isolate state origin. The second column shows the classification
as APEC (blue) or AFEC (green). Columns 3 to 12 represent the antimicrobials tested (AMX =
Amoxicillin; CEF = Ceftiofur; CIP = Ciprofloxacin; ENR = Enrofloxacin; NOR = Norfloxacin; GEN =
Gentamicin; NEO = Neomycin; FFC = Florfenicol; DOX = Doxycycline; TET = Tetracycline), red is
complete resistance, yellow is intermediate resistance, and grey is non-resistant/susceptible. The
last column indicates whether the isolate is MDR (black) or non-MDR (white).
APEC isolates presented high frequency of resistance to amoxicillin (72/109; 66.1%),
followed by ceftiofur (62/109; 56.9%), norfloxacin (52/109; 47.7%), tetracycline (37/109;
33.9%), ciprofloxacin (34/109; 31.2%), florfenicol (28/109; 25.7%), doxycycline (26/109;
23.9%), enrofloxacin (25/109; 22.9%), neomycin (16/109; 14.7%), and gentamicin (3/109;
2.8%). In addition, there was also intermediate resistance for six antimicrobials: en-
rofloxacin (46/109; 42.2%), ciprofloxacin (36/109; 33%), doxycycline (9/109; 8.2%), gen-
tamicin (7/109; 6.4%), ceftiofur (5/109; 4.6%), and amoxicillin (2/109; 1.8%). A total of 32
(29.4%) APEC isolates presented MDR profiles; 40.6% (13/32) were from Paraná, 34.4%
(11/32) were from Rio Grande do Sul, and 25% (8/32) were from Minas Gerais. The most
frequent MDR paerns observed in APEC were β-lactams + fluoroquinolones + tetracy-
clines and β-lactams + fluoroquinolones + aminoglycosides + tetracyclines.
AFEC isolates also presented high frequency of resistance to amoxicillin (13/17;
76.5%), followed by ceftiofur (10/17; 58.8%), tetracycline (8/17; 47.1%), norfloxacin (7/17;
41.2%), neomycin and doxycycline (6/17; 35.3%, each), ciprofloxacin and florfenicol (5/17;
29.4%, each), and finally enrofloxacin (4/17; 23.5%). In addition, there was also interme-
diate resistance for four antimicrobials: ciprofloxacin (5/17; 29.4%), enrofloxacin (4/17;
23.6%), ceftiofur, and amoxicillin (1/17; 5.9%, each). A total of nine (53%) AFEC isolates
presented MDR profiles; 66.7% (6/9) were from Paraná and 33.3% (3/9) were from Rio
Grande do Sul. The most frequent MDR paern observed in AFEC was β-lactams +
aminoglycosides + tetracyclines.
The overall analysis according to the antimicrobial classes demonstrated a high
frequency of resistance to β-lactams (57–68%), fluoroquinolones (23–47%), tetracyclines
Figure 3. Multiple and individual profiles of antimicrobial susceptibility testing in 126 E. coli isolates.
The first column indicates the isolate state origin. The second column shows the classification as APEC
(blue) or AFEC (green). Columns 3 to 12 represent the antimicrobials tested (AMX = Amoxicillin;
CEF = Ceftiofur;
CIP = Ciprofloxacin; ENR = Enrofloxacin; NOR = Norfloxacin; GEN = Gentamicin;
NEO = Neomycin; FFC = Florfenicol; DOX = Doxycycline; TET = Tetracycline), red is complete
resistance, yellow is intermediate resistance, and grey is non-resistant/susceptible. The last column
indicates whether the isolate is MDR (black) or non-MDR (white).
AFEC isolates also presented high frequency of resistance to amoxicillin (13/17; 76.5%),
followed by ceftiofur (10/17; 58.8%), tetracycline (8/17; 47.1%), norfloxacin (7/17; 41.2%),
neomycin and doxycycline (6/17; 35.3%, each), ciprofloxacin and florfenicol (5/17; 29.4%,
each), and finally enrofloxacin (4/17; 23.5%). In addition, there was also intermediate resis-
tance for four antimicrobials: ciprofloxacin (5/17; 29.4%), enrofloxacin (4/17; 23.6%), ceftiofur,
and amoxicillin (1/17; 5.9%, each). A total of nine (53%) AFEC isolates presented MDR pro-
files; 66.7% (6/9) were from Paraná and 33.3% (3/9) were from Rio Grande do Sul. The most
frequent MDR pattern observed in AFEC was β-lactams + aminoglycosides + tetracyclines.
The overall analysis according to the antimicrobial classes demonstrated a high fre-
quency of resistance to
β
-lactams (57–68%), fluoroquinolones (23–47%), tetracyclines
(
25–36%
), and amphenicol (26.5%). The resistance to aminoglycosides was below 20%.
Noteworthy, all AFEC isolates were susceptible to gentamicin.
The results obtained show similarity in resistance of APEC and AFEC isolates. APEC
presented a profile with resistance from none (13/109; 11.9%) to nine (1/109; 0.9%) out of
the ten tested antimicrobials (mean = 3.3; median = 3), while AFEC presented a profile with
resistance from none (1/17; 5.9%) to seven (3/17; 17.6%) out of the ten tested antimicrobials
(mean = 3.8; median = 4). Importantly, AFEC isolates had a higher frequency of MDR (53%)
than APEC (29.4%).
4. Discussion
E. coli is a commensal bacterial species in the enteric microbiota of different domestic
animals, including the main poultry species. However, it can also be an opportunistic
pathogen in many hosts. Of even greater concern are the pathogenic strains of E. coli
adapted to some hosts (poultry, other livestock, and humans), which cause certain diseases
due to a specific arsenal of virulence genes [
1
]. Colibacillosis is an infectious poultry disease
caused by extraintestinal strains of E. coli that leads to a range of clinical manifestations,
including respiratory, systemic, and reproductive infections of chickens in egg and meat
production [22].
Previous studies have demonstrated that E. coli strains of the APEC pathotype are char-
acterized by harboring some very specific genes that encode bacterial proteins/enzymes

Poultry 2025,4, 10 7 of 11
necessary for the infectious process in poultry [
23
,
24
]. These genes are located on chromo-
somes or plasmids, such as ColV and ColBM plasmids [
25
,
26
]. Five main genes (iroN,hlyF,
iutA,iss, and ompT) were further demonstrated in many APEC strains, defining them as
minimal predictors to detect an isolate from this pathotype [
5
]. In the present study, these
same five genes were investigated in 126 isolates to classify them as APEC or AFEC. A total
of 115 (91.3%) isolates were classified as APEC since they presented positive results for
three or more virulence genes analyzed. Even with more rigorous criteria (presence of four
and five of these virulence genes to define pathogenicity), most isolates were considered
APEC: 103 (81.7%) for four and 96 (76.2%) for five genes. The use of these genes to evaluate
pathogenicity in avian E. coli isolates is already well-accepted, but the number of virulence
genes to determine the APEC pathotype has been a recurring topic of discussion, and is
not fully defined [3,5,8].
Furthermore, hlyF and ompT were the most frequent genes in the analyzed isolates,
with 89.7% frequency each. These genes are involved in different virulence mechanisms:
hlyF encodes a hemolysin in the production of external membrane vesicles and autophagy,
while ompT participates in the exterior protection of the bacterial cell against antimicro-
bials [
27
,
28
]. These two genes were also the most frequent in APEC isolates from other
studies with broilers worldwide [
29
–
32
]. In Brazil, there are three studies demonstrating
the high prevalence of these genes in APEC isolates that are circulating in poultry flocks
since 2015 [
3
,
8
,
12
]. On the other hand, iutA gene had the lowest frequency (80.2%) in
comparison with the other four analyzed genes. It is involved in the iron acquisition system
(together with iroN), an essential chemical element for APEC invasion and proliferation
in the host [
31
–
33
]. Other studies also showed the absence of iutA in some APEC iso-
lates [
3
,
29
,
31
,
32
]. The lack of this gene and its protein is compensated by the presence
of other genes and metabolic pathways, since iron acquisition systems in bacteria con-
sist of multiple siderophores (aerobactin, salmochelin, yersiniabactin) and transporters to
sequester iron from the body fluids [34].
In addition, the same five genes evaluated in this study were also detected in APEC
isolates in other previous studies, highlighting their higher frequency than that of other
potential virulence genes (e.g., cvaC,pap,sfa,neuc,ast,vat, and ibeA). This is strong evidence
that factors encoded by these five main virulence genes, many of them carried by plasmids,
are directly associated with the ability to develop colibacillosis in APEC strains [
24
–
26
,
35
].
Therefore, the identification of APEC isolates has been based on the evaluation of this
“pentaplex panel” of minimal predictors [
36
,
37
]. Noteworthy, 86.5% (109/126) samples
were identified as APEC and 13.5% (17/126) as AFEC using these criteria. De Carli et al. [
3
]
and Barbieri et al. [
10
] found a more reduced number of APEC strains (58.7% and 31%,
respectively), but Pilati et al. [
8
] demonstrated a similar frequency (92%) when analyzing
chickens from different Brazilian states. This APEC frequency variation in the studies can
be related to the different bird tissues sampled, geographic location of the farms, and health
and management of the poultry flocks [3,8,10,16,38].
In addition to their pathogenic capacity, many E. coli strains have been characterized
by resistance to antibacterials. Some of these agents (such as penicillin, ciprofloxacin,
streptomycin, sulfamethoxazole, and tetracycline) have been used to treat infections as
well as prophylaxis and metaphylaxis to reduce pathogenic bacteria in poultry flocks [
9
].
Overall, the present study demonstrated the occurrence of E. coli resistance to antibacterials
belonging to the main classes of these antimicrobials already used in poultry practice.
Importantly, resistance to
β
-lactams (57–68%), fluoroquinolones (23–47%), tetracyclines
(25–36%), phenicols (26.5%), and aminoglycosides (<20%) in APEC and also in AFEC
strains was demonstrated. In addition to our study, high levels of resistance to older drugs

Poultry 2025,4, 10 8 of 11
widely used in poultry production have been demonstrated in E. coli isolated from poultry
flocks, as noted in a recent review [22].
The highest percentage of resistance was observed for amoxicillin (68.9%) when it was
compared with the other antimicrobials tested. A previous study reported that 83.3% of the avian
E. coli isolates were resistant to amoxicillin, and only 16.7% to the combination of amoxicillin
with clavulanate [
31
]. It is noteworthy that amoxicillin has been used together with clavulanate
for general therapeutic purposes, since the combination of these antimicrobials has been much
more successful in the treatment of avian infections [
10
,
39
,
40
]. As for the other antibacterials, the
percentages of resistance varied from almost 60% (for ceftiofur) to less than 10% (gentamicin).
Other studies have also demonstrated that APEC isolates are more susceptible to gentamicin
and other antibacterials from this aminoglycosides class [
28
,
41
]. More specifically in Brazil,
studies showed that less than 25% of isolates from chicken treated with antimicrobials were
resistant to gentamicin and neomycin [
42
,
43
]. In addition, florfenicol (amphenicol class) also
showed a low resistance result of 26.2% in the antimicrobial susceptibility test, a rate similar to
other reports [8,11].
This study also reported concerning frequencies of MDR in both APEC (27.5%) and AFEC
(53%) strains. Other surveys in poultry farms have shown the occurrence of MDR in APEC
strains in Brazil [
43
] and worldwide [
6
,
30
,
40
,
41
,
44
]. High levels of MDR APEC strains are
strongly related to specific genes inserted into integrons in the bacteria’s genome [
15
]. When
analyzing the relationship between phenotypic and genotypic resistance, specific plasmid-
linked genes are highly related to resistance to various antimicrobials [
24
,
44
,
45
]. In addition,
the high percentage of MDR in AFEC isolates is noteworthy. AFEC lives in microbiota with a
wide diversity of bacterial species, such as the intestinal tract. In such environments, intense
horizontal transfer of mobile genetic elements (plasmids, transposons, insertion sequences)
occurs between different bacteria, resulting in the widespread transfer of antibiotic resistance
genes too. In fact, commensal E. coli has been used as a sentinel microorganism for monitoring
antimicrobial resistance in different animals and in humans [
46
]. Therefore, it is necessary to
continue searching for antibacterial resistance in field E. coli strains, as well as to identify the
genetic basis for a high resistance bacterial profile.
It is also important to highlight that the present study was conducted with a conve-
nience sample from a specific agro-industrial company in Southern Brazil. Although E. coli
isolates were obtained from poultry farms in different Brazilian states that are relatively
distant, the breeds and genetics of the broilers are the same and the management of the
flocks is almost similar in all poultry farms, with few changes due to climate variations in
the different geographic regions. In addition, only five virulence genes were studied here,
and AMR was tested by phenotypic analysis. The advance of the laboratorial methodolo-
gies has enabled more complex analyses, with the whole-genome sequencing (WGS) of E.
coli isolates, providing much more complete information of virulence and AMR bacterial
genes profiles, as already demonstrated [47,48].
Regardless, the data reported here represent an important advance in the knowledge
of APEC and AFEC isolates circulating in intensive poultry production flocks in Brazil in
recent years. New studies that also include more complete analyses of the genomes of E.
coli isolates will be essential for a better understanding of this concerning pathogen that
is highly frequent in Brazilian and global poultry farming and present a zoonotic risk for
other animals and even humans.
5. Conclusions
In conclusion, a high frequency (86.5%) of APEC isolates was detected in Brazilian
poultry farms. The frequency of APEC isolates varied slightly according to the state of origin
of the samples: 96.8% in Minas Gerais, 85% in Rio Grande do Sul and 81.8% in Paraná. AMR

Poultry 2025,4, 10 9 of 11
to different antibacterial chemicals, mainly to amoxicillin (67.4%) and ceftiofur (57.1%), was
observed in both APEC and AFEC isolates. Furthermore, 32.5% of theE. coli isolates presented
a multidrug resistance (MDR) profile. These findings highlight the importance of constant
monitoring of this pathogenic microorganism in poultry farms and the need for a rational use
of antimicrobials to avoid a further increase in bacterial resistance.
Author Contributions: Conceptualization, C.J.L. and V.R.L.; methodology, C.J.L., P.H.C.H. and J.G.;
validation, C.J.L., P.H.C.H., D.K. and V.R.L.; writing—original draft preparation C.J.L., P.H.C.H., J.G.
and V.R.L.; writing—review and editing, C.J.L., P.H.C.H., D.K., J.G. and V.R.L. All authors have read
and agreed to the published version of the manuscript.
Funding: This study was financed by Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq; process number 303647/2023-0). C.J.L was supported by the Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001. D.K. and V.R.L. were also financially
supported by CNPq (process numbers 351240/2023-3 and 303647/2023-0, respectively).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Acknowledgments: The authors thank the staff of Molecular Diagnosis Laboratory of ULBRA for
their technical support.
Conflicts of Interest: D.K. is a CNPq scholarship holder and develops research projects at the
company Simbios Biotecnologia. V.R.L. is a Professor and also works with R&D at this same company.
The other authors declare that the research was carried out in the absence of any commercial or
financial relationships that could be considered as a potential conflict of interest.
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