Evaluation of the Antimicrobial Susceptibility of Escherichia Coli Strains Isolated from Exhibition Pigeons

Annals of the Academy of Romanian Scientists

Series on Agriculture, Silviculture and Veterinary Medicine Sciences

ISSN 2344-2085

Volume

15

, Number

2

/

2026

54

EVALUATION OF THE ANTIMICROBIAL

SUSCEPTIBILITY OF

ESCHERICHIA COLI

STRAINS

ISOLATED FROM EXHIBITION PIGEONS

Alexandru GLIGOR

12

, Vlad IORGONI

13

, Paula NISTOR

14

,

Ionica IANCU

15

, Viorel HERMAN

16

, Agatha POPESCU

17

Abstract.

Antimicrobial resistance represents one of the major current challenges for

both human and veterinary medicine, with antibiotic use exerting selective pressure that

favors the emergence and dissemination of resistant bacteria. Pigeons (Columba livia

domestica), through their frequent contact with humans, other animal species, and

various sources of contamination in urban and peri-urban environments, may contribute

to

the

circulation

of

antimicrobial-resistant

microorganisms.

Escherichia

coli,

a

commensal bacterium of the avian gastrointestinal tract, is also an important indicator

for antimicrobial resistance surveillance and may include strains with pathogenic and

zoonotic potential. The aim of this study was to evaluate the antimicrobial susceptibility

profiles of Escherichia coli strains isolated from pigeons in Romania. The identified

resistance profiles were analyzed to highlight co-resistance patterns and phenotypic

associations

among

the

antimicrobial

agents

investigated

and

were

descriptively

contextualized against available data on human E. coli isolates. This study contributes to

a better understanding of the role of pigeons as potential reservoirs of antimicrobial-

resistant bacteria and provides relevant data for antimicrobial resistance surveillance at

the animal–human–environment interface within a One Health framework

.

Keywords:

Escherichia

coli

;

antimicrobial

resistance;

pigeons;

co-resistance;

One

Health.

DOI

10.56082/annalsarsciagri.2026.2.54

12

Lecturer PhD. Med. Vet. University of Life Sciences “King Mihai I”, Timisoara, Faculty of

Veterinary Medicine, Romania, e-mail:

alexandru.gligor@usvt.ro

13

PhD student Med. Vet.,

University of Life Sciences “King Mihai I”, Timisoara, Faculty of

Veterinary Medicine, Romania, e-mail:

vlad.iorgoni@usvt.ro

14

Assist.Prof. Ph.D. student Med.Vet.,

University of Life Sciences “King Mihai I”, Timisoara,

Faculty of Veterinary Medicine, Romania, e-mail:

paula.nistor@usvt.ro

15

Assoc.Prof. PhD.Med.Vet.,

University of Life Sciences “King Mihai I”, Timisoara, Faculty of

Veterinary Medicine, Romania, e-mail:

ionica.iancu@usvt.ro

16

Prof.PhD

Med.Vet,

University

of

Life

Sciences

“King

Mihai

I”,

Timisoara,

Faculty

of

Veterinary Medicine, Romania, Corresponding Member of Academy of Romanian Scientists, e-

mail:

viorel.herman@fmvt.ro

17

Prof. PhD Eng.Ec., University of Agronomic Sciences and Veterinary Medicine, Faculty of

Management

and

Rural

Development,

Bucharest,

Romania,

Full

Member

of

Academy

of

Romanian

Scientists,

Corresponding

Member

of

the

Academy

of

Agricultural

and

Forestry

Sciences “ Gheorghe Ionescu-Sisesti”, Bucharest, Romania, e-mail:

agatha_popescu@yahoo.com

Evaluation of the Antimicrobial Susceptibility

of

Escherichia Coli

Strains Isolated from Exhibition Pigeons

55

1.

Introduction

Antimicrobial resistance (AMR) is a major challenge for human and veterinary

medicine.

Antibiotic

exposure

creates

selective

pressure

that

favors

resistant

bacteria

,

and

both

human

medicine

and

animal

production

contribute

to

their

emergence and dissemination [1–5].

The

historical

use

of

antimicrobials

as

growth

promoters

and

their

continued

inappropriate use in some regions have reinforced the need for prudent antimicrobial

use and alternative preventive approaches [3–7].

Pigeons (Columba livia domestica) are of particular interest in AMR surveillance

because

they

live

in

close

contact

with

humans,

domestic

animals,

wild

birds,

contaminated water, animal feces, and urban waste [8–11]. These ecological interfaces

may facilitate acquisition, carriage, and environmental dissemination of resistant

microorganisms.

Escherichia coli

is a common intestinal commensal of birds and mammals and an

important indicator organism for AMR surveillance [12-16]. Avian pathogenic E

. coli

(APEC), enteropathogenic E. coli (EPEC), and Shiga toxin-producing

E. coli

(STEC)

are relevant because avian strains may share virulence and resistance traits with human

extraintestinal pathogenic

E. coli

, raising One Health concerns [17-25].

Previous studies from Romania have also documented antimicrobial-resistant

E. coli

in

avian and other animal hosts, further supporting the relevance of integrated AMR

surveillance

[26–29]

.

In Romania, domestic pigeons are maintained for racing, exhibitions, ornamental

breeding, and recreational purposes, often in urban or peri-urban settings. Despite their

close interaction with humans and other birds, systematic information on antimicrobial

susceptibility

in

these

populations

remains

limited.

Studies

conducted

in

other

European countries have demonstrated that domestic pigeons may carry

E. coli

isolates

displaying resistance to several antimicrobial classes

[30,31]

.

The aim of the present study was to evaluate antimicrobial susceptibility profiles of

E.

coli

strains isolated from Romanian pigeons during 2024–2025. Co-resistance patterns,

clustering, MDR phenotypes, and MIC distributions were assessed, and the results were

descriptively contextualized against available human

E. coli

resistance data within a

One Health framework.

2.

Materials and methods

2.1 Study Design

The present study was designed as a cross-sectional observational investigation

conducted during 2024–2025. The study population consisted of domestic pigeons

(

Columba livia domestica

) raised for racing, recreational purposes, or breeding in

several regions of Romania.

Alexandru

Gligor

, Vlad

Iorgoni

, Paula

Nistor

,

Ionica

Iancu

, Viorel

Herman

, Agatha

Popescu

56

Samples were collected during the National Exhibition organized by the Fauna

Banatului Association in Timișoara. The pigeons included in the study originated

from lofts located in the counties of Timiș, Arad, Bihor, Hunedoara, Caraș-

Severin, Gorj, and Alba and were presented at the exhibition. The flocks were

selected based on breeder availability, previous collaboration with the official

veterinarian, and geographical distribution, with the aim of ensuring the broadest

possible representation of different regions of the country.

The inclusion criteria comprised clinically healthy birds over six months of age

that had not received antimicrobial treatment during the two weeks preceding

sample

collection.

Birds

showing

clinical

signs

of

disease

or

for

which

the

antimicrobial treatment history was unknown were excluded from the study.

Oropharyngeal and cloacal samples were collected using sterile swabs placed in

Deltalab

transport

systems

containing

Amies

medium.

Bacteriological

identification

was

performed

by

inoculating

the

samples

onto

ChromoBio

Coliform agar, and the resulting pure cultures were preserved using Microbank

systems and stored at −80 °C until further analyses were performed.

A total of 134

Escherichia coli

strains were included in the final analysis.

The sample size was determined pragmatically based on field accessibility and the

logistical feasibility of the study and was considered sufficient for the planned

statistical analyses. Strains were selected based on colony morphology, sample

type, and flock of origin, with the aim of reducing the overrepresentation of

particular isolates and maximizing epidemiological diversity within the practical

constraints of bacterial isolation and characterization.

2.2.

Determination of the Minimum Inhibitory Concentration (MIC)

The antibiotics included in the susceptibility testing were selected based on their

documented and routine use in veterinary medicine, particularly for the treatment

of bacterial infections in domestic pigeons in Romania. All pigeons included in

the present study were domestic birds raised for breeding, racing competitions, or

ornamental purposes. They were maintained under controlled conditions and

housed in pigeon lofts or aviaries. None of the birds included in the study

originated

from

wild

populations

or

were

free-ranging.

The

antibiotic

panel

included antimicrobial agents relevant both to avian clinical practice and from a

public health perspective. The phenotypic expression of resistance was evaluated

by determining minimum inhibitory concentration (MIC) values with reference to

the Clinical and Laboratory Standards Institute (CLSI) framework for dilution

susceptibility

testing

[38].

Breakpoints

were

established

according

to

CLSI

recommendations

[32]

and

were

compared,

where

applicable,

with

Evaluation of the Antimicrobial Susceptibility

of

Escherichia Coli

Strains Isolated from Exhibition Pigeons

57

epidemiological cutoff values (ECOFFs) defined by the European Committee on

Antimicrobial Susceptibility Testing (EUCAST) [33]. For antimicrobial agents for

which species-specific interpretive criteria were unavailable, additional criteria

were derived from relevant scientific literature [30,31,34].

Prior to testing, bacterial strains stored at −80 °C were resuspended in 3 mL of

cation-adjusted Mueller–Hinton broth (CAMHB) and incubated for 18–24 h at 37

°C. Antimicrobial susceptibility testing was performed using 96-well microtiter

plates. All wells, except those in the first column, were loaded with 90 μL of

CAMHB.

Stock solutions of the tested antibiotics (Merck KGaA, Darmstadt,

Germany) were prepared at a concentration of 1,024 μg/mL in accordance with

CLSI recommendations

[32]

.

Amoxicillin and the amoxicillin–clavulanic acid combination were prepared at a

2:1 ratio (pH 7.2; 0.01 mol/L), while imipenem was dissolved in phosphate buffer

(pH

6;

0.1

mol/L).

Doxycycline,

neomycin,

tylosin,

and

vancomycin

were

dissolved

in

distilled

water.

For

preparation

of

the

trimethoprim–

sulfamethoxazole mixture (1:19 ratio), sulfamethoxazole was dissolved in hot

water with a few drops of 2.5 mol/L NaOH, while trimethoprim was dissolved in

distilled

water

containing

0.05

mol/L

HCl.

Enrofloxacin

was

prepared

by

dissolving the compound in distilled water with the addition of 100 μL of 1 mol/L

NaOH. Florfenicol was dissolved in distilled water with the addition of 100 μL of

95% ethanol to improve solubility.

A dilution with a concentration of 512 μg/mL was prepared from these solutions,

and 180 μL was dispensed into the first column of the microtiter plates. Two-fold

serial

dilutions

were

subsequently

performed

across

the

plate,

with

90

μL

removed from the final well so that each well contained a final volume of 90 μL.

Bacterial

suspensions

were

adjusted

to

a

0.5

McFarland

standard

using

a

nephelometer, and 10 μL of bacterial suspension was inoculated into 90 μL per

well, as documented in the laboratory record. The final inoculum concentration

was not recorded in the available documentation and is therefore acknowledged as

a methodological limitation [38].

MIC values were determined using the automated Sensititre™ SWIN™ MIC

Reader together with VIZION System software, version 3.4. Escherichia coli

ATCC 25922 was used as the quality-control strain. The resulting MIC values

were interpreted against the specified international criteria.

MIC

₅₀

and

MIC

₉₀

values

were

calculated

as

the

50th

and

90th

percentiles,

respectively, of the MIC value distribution for all tested isolates. These values

represent

the

minimum

inhibitory

concentrations

at

which

50%

and

90%,

respectively,

of

the

investigated

bacterial

isolates

were

inhibited.

MIC

distributions were obtained from the complete dataset of 134

Escherichia coli

Alexandru

Gligor

, Vlad

Iorgoni

, Paula

Nistor

,

Ionica

Iancu

, Viorel

Herman

, Agatha

Popescu

58

isolates tested for each antibiotic, and the percentiles were calculated using the

base functions of R software. These parameters provide an overall assessment of

antimicrobial susceptibility trends within the bacterial population analyzed.

2.3.

Statistical Analysis

Statistical analyses were performed in R version 4.2.2 [35]. The analyses reported

here

focused

on

descriptive

susceptibility

summaries,

binary

resistance

associations, clustering, and exploratory modeling.

Resistance

data

were

binarized

(resistant

=

1;

susceptible

=

0).

Pairwise

associations were evaluated using the phi coefficient (φ), and heatmaps were

generated with corrplot and pheatmap. Antimicrobial variables with zero variance

were excluded from phi-coefficient calculations because associations cannot be

estimated

when

all

isolates

have

the

same

binary

outcome.

Accordingly,

trimethoprim–sulfamethoxazole (potentiated sulfonamide), for which no resistant

isolates

were

detected,

was

excluded

from

the

phi

correlation

matrix.

Agglomerative hierarchical clustering was explored using Jaccard dissimilarities.

PCA visualization was performed with factoextra.

Co-resistance networks were constructed with igraph and ggraph, with edge

weights

representing

the

frequency

of

simultaneous

resistance.

Decision-tree

models (rpart) were used exploratorily to identify predictors of MDR phenotypes.

Uncertainty

around

MDR

prevalence

was

evaluated

using

a

Monte

Carlo

simulation

with

10,000

iterations,

in

which

the

initial

resistance

rates

were

randomly varied by ±20%. The resulting distribution was used to estimate the

mean, median, standard deviation, and 95% percentile interval of the simulated

distribution (not a confidence interval for observed prevalence).

2.4. Definitions of Resistance Categories

The definitions proposed by Magiorakos et al. [36] were used as a conceptual

framework. MDR denotes acquired non-susceptibility in at least three relevant

antimicrobial

categories.

Intrinsically

inactive

agents,

including

tylosin

and

vancomycin for E. coli, were not considered informative for acquired-resistance

classification. XDR and PDR were not reported as confirmed phenotypes because

the antimicrobial panel did not provide sufficiently complete category coverage

for robust classification according to the Magiorakos framework.

3. Results and Discussions

Evaluation of the Antimicrobial Susceptibility

of

Escherichia Coli

Strains Isolated from Exhibition Pigeons

59

3.1. Characteristics of the Study

A

total

of

660

samples

(330

cloacal

and

330

oropharyngeal

samples)

were

collected from 330 domestic pigeons.

E. coli was identified in 328/660 samples

(49.7%). From these positive samples, 134 isolates representing 20 flocks were

selected

for

antimicrobial

susceptibility

testing

according

to

the

predefined

selection criteria.

The majority of isolates originated from Timiș County (52.2%). Most strains

(50.0%) were isolated from racing pigeons, while the majority of the birds

investigated

were

young

adults

(32.8%)

and

adult

birds

used

for

breeding

(28.4%).

Most

pigeon

breeders

(47.8%)

maintained

medium-sized

flocks

comprising between 51 and 100 birds.

Figure 1.

Geographical distribution of the sampling locations included in the study. The map

highlights the seven counties in western Romania where biological samples were collected: Timiș,

Arad, Bihor, Hunedoara, Caraș-Severin, Gorj, and Alba.

A

total

of

134

E.

coli

isolates

representing

20

flocks

from

seven

western

Romanian counties were included in susceptibility testing.

Birds were used mainly for racing, ornamental, meat-production, or breeding

purposes, with ages ranging from 6 months to over 4 years. Table 1 summarizes

their distribution by county and primary purpose of rearing.

Table 1.

Distribution of E. coli isolates according to county and primary purpose of pigeon

rearing.

Alexandru

Gligor

, Vlad

Iorgoni

, Paula

Nistor

,

Ionica

Iancu

, Viorel

Herman

, Agatha

Popescu

60

County

Racing pigeons n (%)

Meat production n (%)

Ornamental n (%)

Timiș

26 (19.4%)

21 (15.7%)

23 (17.2%)

Arad

11 (8.2%)

0 (0.0%)

13 (9.7%)

Bihor

3 (2.2%)

0 (0.0%)

5 (3.7%)

Hunedoara

0 (0.0%)

5 (3.7%)

0 (0.0%)

Caraș-Severin

4 (3.0%)

0 (0.0%)

Gorj

10 (7.5%)

0 (0.0%)

Alba

13 (9.7%)

0 (0.0%)

3.2. Resistance Correlations and Cluster Analysis

Phi coefficient analysis revealed varying degrees of association among antibiotic

resistance

profiles.

Using

the

heatmap

representation

(Figure

2),

stronger

correlations were identified that may indicate the phenotypic co-occurrence of

resistance

traits.

Although

these

correlations

do

not

demonstrate

a

causal

relationship or the presence of underlying genetic mechanisms, they may reflect

phenomena such as co-selection, co-resistance, or cross-resistance, for example,

as a result of resistance genes being located on the same mobile genetic element

or the concurrent use of multiple antimicrobial agents.

The

strongest

positive

correlation

was

observed

between

ceftriaxone

and

imipenem

(φ

=

0.70).

Other

positive

associations

included

florfenicol

and

neomycin (φ = 0.36) and amoxicillin–clavulanic acid and enrofloxacin (φ = 0.31).

The most pronounced negative correlation was observed between amoxicillin and

colistin (φ = −0.13). Trimethoprim–sulfamethoxazole was not included in the phi

analysis because no resistant isolates were detected, resulting in a zero-variance

binary variable for which φ is undefined.

Cluster analysis identified three main groups, within which different antimicrobial

agents were predominant in terms of resistance.

In Cluster 1, neomycin showed the highest frequency of resistance (95.2%),

whereas Cluster 2 was characterized by a predominance of doxycycline resistance

(75.8%). In Cluster 3, neomycin was again the predominant antibiotic, with a

resistance frequency of 92.3%.

Evaluation of the Antimicrobial Susceptibility

of

Escherichia Coli

Strains Isolated from Exhibition Pigeons

61

Figure 2.

Heatmap of phi coefficients for pairwise antibiotic resistance profiles among

Escherichia coli strains (n = 134) isolated from pigeons. Trimethoprim–sulfamethoxazole was

excluded because the resistance variable had zero variance (0 resistant isolates).

3.3. Co-Resistance Network Analysis

Network Graph Analysis revealed the relationships and frequency of connections

among antibiotic resistance profiles. Within the network, line thickness represents

the

frequency

of

co-resistance

between

pairs

of

antibiotics.

Antibiotics

are

represented by light blue circles, with their sizes proportional to the number of

resistant isolates associated with each antimicrobial agent.

The most frequent associations were observed among neomycin, doxycycline, and

florfenicol, indicating frequent phenotypic co-occurrence of resistance to these

antimicrobial agents.

3.4. Predictive Modeling and Simulation

The

decision

tree-based

model

enabled

the

prediction

of

multidrug-resistant

(MDR) Escherichia coli strains. The model results demonstrated that resistance to

certain antibiotics represents an important predictor of whether a strain belongs to

the

MDR

category.

In

particular,

resistance

to

neomycin,

doxycycline,

and

amoxicillin

emerged

as

important

predictors

of

the

MDR

phenotype

in

this

exploratory model.

Alexandru

Gligor

, Vlad

Iorgoni

, Paula

Nistor

,

Ionica

Iancu

, Viorel

Herman

, Agatha

Popescu

62

To assess the potential variability in the prevalence of MDR phenotypes, a Monte

Carlo

simulation

was

performed,

in

which

the

initial

resistance

rates

were

randomly varied by ±20%. The estimated mean prevalence of MDR strains was

62.1%, while the median was 61.9%, indicating an approximately symmetrical

distribution of the results.

The standard deviation was 7.2%, suggesting that the occurrence rate of MDR

strains

generally

ranged

between

54.9%

and

69.3%.

The

95%

simulation

percentile

interval

ranged

from

47.7%

to

75.3%,

reflecting

the

variability

generated under the specified simulation assumptions; this interval should not be

interpreted as a confidence interval for the observed prevalence.

3.5. Prevalence of Antimicrobial Resistance and MIC Values

Based on the determined minimum inhibitory concentration (MIC) values, a

frequency table (Figure 3) was generated to summarize the results obtained,

including the clinical breakpoint for each antibiotic. MIC

₅₀

and MIC

₉₀

values

were also calculated for the bacterial population investigated.

Figure 3..

Frequency distribution of minimum inhibitory concentration (MIC) values for

Escherichia coli

strains (n = 134) isolated from pigeons and tested against antibiotics for which

clinical breakpoints have been established.

Among

the

antimicrobial

agents

tested,

ceftriaxone

and

imipenem

exhibited

MIC

₉₀

values below the established clinical breakpoints, indicating that at least

90% of the tested isolates had MIC values below the corresponding clinical

breakpoints. Analysis of the MIC

₅₀

values showed that amoxicillin, amoxicillin–

clavulanic acid, ceftriaxone, colistin, enrofloxacin, and imipenem had values

below the established clinical breakpoints. These findings indicate that at least

half of the isolates retained susceptibility to these antimicrobial agents.

Evaluation of the Antimicrobial Susceptibility

of

Escherichia Coli

Strains Isolated from Exhibition Pigeons

63

When

the

results

were

compared

with

the

epidemiological

cutoff

values

(ECOFFs)

established

by

the

European

Committee

on

Antimicrobial

Susceptibility Testing (EUCAST), it was observed that, for ceftriaxone, colistin,

and imipenem, at least half of the investigated isolates were most likely classified

as wild-type strains. This comparison was performed to distinguish acquired

resistance (non-wild-type strains) from intrinsic susceptibility, independently of

the clinical breakpoints used in medical practice. By concurrently evaluating

phenotypic

resistance

and

ECOFF-based

wild-type

status,

a

more

detailed

assessment of emerging antimicrobial resistance trends was sought, including

among isolates that are not yet clinically classified as resistant. Such information

may support the early detection of emerging resistance trends and epidemiological

surveillance within a One Health framework.

Detailed MIC values, together with the individual data corresponding to each

bacterial isolate, are provided in the Supplementary Material.

The

highest

resistance

levels

were

recorded

for

neomycin

(76.1%),

an

aminoglycoside antibiotic, and florfenicol (72.4%), a member of the phenicol

class. In contrast, no phenotypically resistant isolates were identified for the

trimethoprim–sulfamethoxazole

combination,

representing

a

potentiated

sulfonamide. The lowest resistance levels were observed for ceftriaxone (0.7%), a

third-generation cephalosporin, and imipenem (1.5%), a carbapenem antibiotic.

3.6. Multidrug Resistance Patterns

The occurrence of multidrug-resistant (MDR) phenotypes was evaluated, while

the applicability of XDR and PDR classifications was assessed according to the

Magiorakos framework.

Based on the operational MDR classification applied in the present analysis, 85 of

134

isolates

(63.4%)

were

assigned

an

MDR

phenotype.

XDR

and

PDR

frequencies were not reported as confirmed findings because the antimicrobial

panel

did

not

provide

sufficiently

complete

category

coverage

for

robust

classification according to the Magiorakos framework. The absence of resistance

to

trimethoprim–sulfamethoxazole

further

precluded

a

confirmed

PDR

designation within the tested panel.

3.7. Comparison with Human Antibiotic Resistance Data

The resistance profiles of

Escherichia coli

isolates obtained from pigeons were

compared with antibiotic resistance data from human clinical isolates provided by

the Timișoara Public Health Directorate, in accordance with the study objective of

investigating

potential

overlaps

in

resistance

patterns

within

the

One

Health

framework (Figure 4).

Alexandru

Gligor

, Vlad

Iorgoni

, Paula

Nistor

,

Ionica

Iancu

, Viorel

Herman

, Agatha

Popescu

64

Figure 4.

Comparison of the resistance profiles of

Escherichia coli

strains isolated from pigeons

with antibiotic resistance data from the human public health system

.

Regarding β-lactam antibiotics, lower resistance levels were observed among

pigeon isolates compared with human clinical strains; these differences may

reflect

distinct

antimicrobial-exposure

patterns

and

the

differing

sampling

frameworks of veterinary and human surveillance. An exception was imipenem,

for which a resistance rate of 1.5% was identified among pigeon isolates, whereas

no resistance was recorded in the corresponding human medicine data. The

detection of imipenem resistance in a small proportion of pigeon isolates warrants

further

investigation,

particularly

through

molecular

characterization

of

the

underlying

resistance

determinants.

Resistance

to

fluoroquinolones

showed

a

relatively similar distribution between isolates of veterinary origin and those from

human

medicine.

For

the

trimethoprim–sulfamethoxazole

combination

(potentiated sulfonamide), no resistant strains were identified among the pigeon

isolates. In contrast, a high resistance frequency was observed for neomycin, the

aminoglycoside included in the pigeon antimicrobial panel.

3.8.

Discussion

of

Antimicrobial

Resistance

in

Escherichia

coli

Strains

Isolated from Pigeons

The present study identified a high burden of antimicrobial resistance among 134

E. coli isolates from domestic pigeons. Based on the operational classification

applied in the present analysis, 63.4% (85/134) of isolates exhibited an MDR

phenotype.

XDR

and

PDR

frequencies

were

not

reported

because

the

antimicrobial panel did not provide sufficiently complete category coverage for

robust classification according to the Magiorakos framework.

Karim et al. reported a lower MDR prevalence (23.8%) in pigeon

E. coli

from

Bangladesh, with frequent resistance to amoxicillin, ampicillin, erythromycin, and

tetracycline

[37]

.

Differences

from

the

present

study

may

reflect

regional

antimicrobial-use practices, sampling design, husbandry, and veterinary oversight.

Evaluation of the Antimicrobial Susceptibility

of

Escherichia Coli

Strains Isolated from Exhibition Pigeons

65

The absence of reliable treatment records in the investigated flocks prevents direct

attribution of the observed phenotypes to previous antimicrobial exposure.

The MIC results further characterized the susceptibility profile. MIC90 values for

ceftriaxone

(0.25

μg/mL)

and

imipenem

(1

μg/mL)

remained

below

clinical

breakpoints, whereas MIC50 values indicated retained susceptibility in at least

half of isolates for several agents.

A Belgian study reported enrofloxacin MIC

₅₀

<0.25 μg/mL and MIC

₉₀

32 μg/mL

[34]

, compared with 0.125 and 8 μg/mL,

respectively,

in

the

present

dataset.

Recent

data

from

Hungary

have

also

demonstrated heterogeneous antimicrobial susceptibility profiles among

E. coli

isolates from domestic pigeons, emphasizing the importance of geographical

differences in antimicrobial exposure and resistance epidemiology

[30]

.

Resistance

frequencies

differed

substantially

among

agents.

Neomycin

and

florfenicol

showed

the

highest

resistance

levels,

whereas

ceftriaxone

and

imipenem

remained

low.

No

resistance

was

detected

to

trimethoprim–

sulfamethoxazole. Comparable international studies have reported heterogeneous

profiles, emphasizing geographical variation in antimicrobial selection pressure

and management practices [18,31].

Correlation and clustering analyses identified structured co-resistance patterns.

The strongest positive association occurred between ceftriaxone and imipenem (φ

≈ 0.7), with additional positive associations observed between florfenicol and

neomycin

and

between

amoxicillin–clavulanic

acid

and

enrofloxacin.

Trimethoprim–sulfamethoxazole was excluded from the phi analysis because all

isolates were susceptible, making the binary resistance variable invariant and the

phi

coefficient

undefined.

Such

phenotypic

associations

do

not

demonstrate

shared genetic mechanisms but may be compatible with co-selection, linked

resistance determinants, altered permeability, or multidrug efflux systems.

Cluster and network analyses consistently highlighted neomycin, doxycycline,

and florfenicol. High doxycycline resistance may reflect established tetracycline-

resistance mechanisms, including efflux and ribosomal protection, while the broad

distribution of aminoglycoside resistance reduced the discriminatory value of

neomycin across clusters. Molecular studies are required to determine whether

mobile genetic elements contribute to these patterns.

Comparison with human surveillance data showed lower resistance among pigeon

isolates for several β-lactams, while some resistance trends were similar across

sectors. These descriptive parallels should not be interpreted as evidence of

transmission because the veterinary and human datasets differ in sampling design,

surveillance methodology, and antimicrobial panels. Nevertheless, they support

integrated One Health surveillance.

Alexandru

Gligor

, Vlad

Iorgoni

, Paula

Nistor

,

Ionica

Iancu

, Viorel

Herman

, Agatha

Popescu

66

The study has several limitations. Sampling was pragmatic rather than strictly

randomized;

antimicrobial-use

histories

were

incomplete;

the

cross-sectional

design does not support temporal or causal inference; and resistance mechanisms

were inferred from phenotypic data without PCR or whole-genome sequencing.

These factors limit generalizability and mechanistic interpretation. The available

laboratory documentation did not record the final broth-microdilution inoculum

concentration. In addition, incomplete antimicrobial-category coverage precluded

robust classification of XDR and PDR phenotypes.

Future

studies

should

combine

longitudinal

sampling

with

standardized

antimicrobial-use

records

and

genomic

characterization

of

resistance

determinants. Harmonized veterinary and human surveillance would also improve

comparison across sectors and help clarify the epidemiological role of domestic

pigeons.

Overall, the findings support continued AMR monitoring in pigeon populations

and antimicrobial stewardship among breeders. The substantial MDR proportion

observed under the operational classification, together with co-resistance patterns

and the close human–animal–environment interface, supports further investigation

within a One Health framework.

4. Conclusions

Among E. coli strains isolated from domestic pigeons in western Romania, 63.4%

(85/134)

were

assigned

an

MDR

phenotype

according

to

the

operational

classification applied in the present analysis. XDR and PDR were not reported

because the antimicrobial panel did not provide sufficiently complete category

coverage

for

robust

classification.

Co-occurrence

of

resistance

traits

was

particularly evident for neomycin, doxycycline, and florfenicol, although the

molecular basis of these associations was not investigated.

The results indicate a need for periodic AMR surveillance, prudent antimicrobial

use,

improved

treatment

records

in

pigeon

holdings,

and

molecular

characterization of resistance determinants. Because the study was cross-sectional

and sampling was not strictly randomized, the findings should be interpreted as

evidence from the investigated population rather than as national prevalence

estimates.

Integration of veterinary, human, and environmental AMR data within a One

Health framework would strengthen surveillance and help assess the potential role

of pigeons as reservoirs and disseminators of resistant

E. coli

.

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