animals

Article
Epidemiological Characterization of Isolates of
Salmonella enterica and Shiga Toxin-Producing Escherichia coli
from Backyard Production System Animals in the Valparaíso
and Metropolitana Regions
Constanza Urzúa-Encina 1,2 , Bastián Fernández-Sanhueza 1,2,3 , Erika Pavez-Muñoz 1,2 ,
Galia Ramírez-Toloza 1,2 , Mariela Lujan-Tomazic 4,5 , Anabel Elisa Rodríguez 4 and Raúl Alegría-Morán 3, *

1 Departamento de Medicina Preventiva Animal, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de

Chile, Santa Rosa 11735, La Pintana, Santiago 8820808, Chile; constanza.urzua23@gmail.com (C.U.-E.);
bastian.fernandez.s@ug.uchile.cl (B.F.-S.); isabelpm95@gmail.com (E.P.-M.); galiaram@uchile.cl (G.R.-T.)
2 Laboratorio Centralizado de Investigación Veterinaria, Facultad de Ciencias Veterinarias y Pecuarias,
Universidad de Chile, Santa Rosa 11735, La Pintana, Santiago 8820808, Chile
3 Escuela de Medicina Veterinaria, Sede Santiago, Facultad de Recursos Naturales y Medicina Veterinaria,
Universidad Santo Tomás, Ejercito Libertador 146, Santiago 8370003, Chile
4 Instituto de Patobiología Veterinaria, Instituto Nacional de Tecnologías Agropecuarias, Consejo Nacional de
Investigaciones Científicas y Técnicas, Av. de los Reseros y Nicolás Repetto s/n, Hurlingham,
Buenos Aires 1686, Argentina; tomazic.mariela@inta.gob.ar (M.L.-T.); rodriguez.anabel@inta.gob.ar (A.E.R.)
5 Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Av. Junín 954,
Buenos Aires C1113 AAD, Argentina
* Correspondence: ralegria2@santotomas.cl; Tel.: +56-998223891

Simple Summary: This study aimed to investigate the prevalence and risk factors for Salmonella enterica
Citation: Urzúa-Encina, C.; and Shiga toxin-producing Escherichia coli (STEC) in backyard production systems (BPS) from central
Fernández-Sanhueza, B.; Chile. BPS were determined as the epidemiologic unit, collecting, for every sampled PS, cloacal or
Pavez-Muñoz, E.; Ramírez-Toloza, G.;
rectal swabs, which were then analyzed by bacterial culture and confirmed by conventional PCR. A
Lujan-Tomazic, M.; Rodríguez, A.E.;
positivity rate of 4.17% was estimated among BPS for S. enterica in the Metropolitana region, while
Alegría-Morán, R. Epidemiological
no positive samples were found in the Valparaíso region. For STEC, a positivity rate among BPS of
Characterization of Isolates of
11.76% in the Metropolitana region and 18.52% in the Valparaíso region was estimated. The study
Salmonella enterica and Shiga
Toxin-Producing Escherichia coli from
also identifies different antimicrobial phenotypical resistance profiles in both S. enterica and STEC,
Backyard Production System including multiresistant strains, considered critically important under the One Health approach.
Animals in the Valparaíso and The presence of ruminants inside BPS was identified as a factor that raises the risk of positivity
Metropolitana Regions. Animals 2023, for STEC and S. enterica/STEC. The study highlights the need for improved biosecurity measures
13, 2444. https://doi.org/10.3390/ and education regarding zoonotic agents in BPS. The findings of this study are of high importance,
ani13152444 providing evidence to policymakers and stakeholders to develop strategies to reduce the risk of
Academic Editor: Laila Darwich transmission of zoonotic agents from BPS to humans and other animals. BPS managed exclusively
by women were shown to be at a greater risk for S. entarica/STEC positivity compared to men
Received: 12 May 2023
of the family, emphasizing the need to implement and deliver training to women to reduce the
Revised: 25 June 2023
current consequences of the gender gap and its potential impact on this animal and human neglected
Accepted: 29 June 2023
population in Chile.
Published: 28 July 2023

Abstract: Backyard production systems (BPS) are distributed worldwide, rearing animals recognized
as reservoirs of Salmonella enterica and Shiga toxin-producing Escherichia coli (STEC), both zoonotic
Copyright: © 2023 by the authors. pathogens. The aim of this study was to characterize isolates of both pathogens obtained from animals
Licensee MDPI, Basel, Switzerland. raised in BPS from two central Chile regions. The presence of pathogens was determined by bacterial
This article is an open access article culture and confirmatory PCR for each sampled BPS, calculating positivity rates. Multivariate logistic
distributed under the terms and regression was used to determine risk factors. Additionally, phenotypic antimicrobial resistance was
conditions of the Creative Commons determined. A positivity rate of 2.88% for S. enterica and 14.39% for STEC was determined for the
Attribution (CC BY) license (https:// complete study region (Valparaíso and Metropolitana regions). Risk factor analysis suggests that the
creativecommons.org/licenses/by/
presence of ruminants (OR = 1.03; 95% CI = 1.002–1.075) increases the risk of STEC-positive BPS, and
4.0/).

Animals 2023, 13, 2444. https://doi.org/10.3390/ani13152444 https://www.mdpi.com/journal/animals

Animals 2023, 13, 2444 2 of 15

the presence of ruminants (OR = 1.05; 95% CI = 1.002–1.075) and the animal handlers being exclusively
women (OR = 3.54; 95% CI = 1.029–12.193) increase the risk for S. enterica/STEC positivity. Eighty
percent of S. enterica isolates were multidrug resistant, and all STEC were resistant to Cephalexin.
This study evidences the circulation of multidrug-resistant zoonotic bacterial strains in animals kept
in BPS and the presence of factors that modify the risk of BPS positivity for both pathogens.

Keywords: Salmonella enterica; STEC; risk factors; antimicrobial resistance; positivity rate; backyard
production system; One Health

1. Introduction
Salmonella enterica and Shiga toxin-producing Escherichia coli (STEC) are zoonotic,
Gram-negative bacilli belonging to the Enterobacteriaceae family [1–4]. Transmission for
both pathogens occur via the oral–fecal route, by direct or indirect contact with infected
animals, fomites, or contaminated foods [2,5–10]. They can be present in various reser-
voirs, some of which are commonly found in rural households, such as poultry, pigs, and
ruminants [11–18]. Both Enterobacteriaceae can cause severe, life-threatening clinical disease
in humans, particularly in the at-risk population, which includes children under 5 and
10 years old, pregnant women, elders, and immunocompromised patients [2,19–24].
In developed countries, S. enterica and STEC are the second and third leading causes
of foodborne disease (FBD) outbreaks, respectively [8,9,25], while in Chile, they are the first
and fifth causes, respectively [26,27].
Backyard production systems (BPS) correspond to small-scale agricultural systems
with extensive or semi-intensive characteristics, being majorly present in rural and low-
income areas [28–30]. Most of them are mixed systems, which perform forestry, agriculture
and/or livestock farming, but commonly none of these are the main activity or source
of income [16,29,31]. Animals raised in BPS (commonly poultry and swine) tend to have
low acquisition and maintenance costs, are of small size, and have short productive cy-
cles, which facilitates their availability for domestic consumption, sale, or exchange when
needed [29,32–34]. In Chile, BPS are mainly managed by women and about 60% of own-
ers are over 55 years old, which increases the likelihood of suffering comorbidities that
lead to immunosuppression [30,35,36]. Therefore, most owners are considered in the
at-risk population. Additionally, animal and human populations that reside in BPS are
frequently neglected.
BPS commonly have poor or no veterinary attention [31], which implies a lack of ade-
quate diagnosis and treatment of diseased animals. Moreover, an absence of production and
sanitary protocols, and knowledge regarding zoonotic agents present in animals [16,33,37],
allows pathogen maintenance and transmission in these systems. Infections with these
pathogens in animals are often asymptomatic and their shedding is intermittent, which
facilitates their persistence and spread to the environment [14,24,38]. In addition, humans
and animals from different species, ages, health status, and even BPS interact in these
landscapes [16,22,33].
Alegria-Moran et al. [16] described a prevalence of S. enterica in BPS of 8.3% in the
Metropolitana region and of 6.6% in the Valparaíso region in Chile. Additionally, they
reported different factors that increase the risk of S. enterica positivity in BPS, including
breeding different bird species (odds ratio (OR) = 1.04; 95% CI: 1.01–1.07), carrying out
mixed production activities (OR = 5.35; 95% CI: 1.2–27.6), and obtaining replacement
animals from external sources (OR = 5.19, 95% CI: 1.4–20.5). On the other hand, STEC
prevalence ranging between 0 to 72% has been reported in cattle in different countries [13].
In central Chile, positivity rates of 17% in cattle and 1% in slaughtered pigs were reported
for STEC [39].
It is known that the emergence and dissemination of antimicrobial resistance (AMR)
in bacteria is considered one of the major threats to public health, affecting human, animal,
Animals 2023, 13, 2444 3 of 15

and environmental health, and, therefore, being identified as one of the major issues
for One Health [40,41]. In Chile, information concerning antimicrobial (AM) usage in
BPS is scarce. Pavez-Muñoz et al. [31] found, for the first time, AMR in STEC strains
obtained from BPS, while also evaluating factors associated with AM usage in BPS from the
Metropolitana region, which included recognition of sick animals, presence of neighboring
poultry and/or swine BPS, visits of veterinary officials, and close contact between animal
species present within BPS. This study also found that AMs were commonly administered
without prescription or control overdose and frequency, increasing the probability of
generating AMR and residues in derived products [42]. Prior studies from central Chile
reported 30 S. enterica strains isolated from backyard animals, with 11 of them showing
either single drug resistance (SDR) or multidrug resistance (MDR) [43].
Considering the absence of biosecurity measures, the highly variable sanitary condi-
tions, the lack of specific knowledge regarding zoonotic pathogens, the emergence of AMR,
and the previously stated background, this is also in frame with the One Health concept,
which, among other issues, concerns antimicrobial resistance. The aim of this study was to
epidemiologically characterize the S. enterica and STEC isolates obtained from BPS in the
Valparaiso and Metropolitana regions of Chile.

2. Materials and Methods

A cross-sectional study was carried out comprising BPS located in the Metropoli-
tana and Valparaíso regions, recording data concerning sample processing (results of
microbiological characterization), epidemiological survey responses (epidemiological char-
acterization), and georeference for each sampled BPS. This also included all data acquired
on isolated and confirmed strains of S. enterica and STEC (with a total of 5 and 37 isolates,
respectively).
In Chile, BPS total over 150 thousand producers; in terms of animal population, it is
estimated that they contain over 3.7 million poultry species and 400 thousand pigs, most of
them concentrated in the studied regions [44].

2.1. Sample Size Calculation

A stratified random sampling with proportional allocation was carried out in the
above-mentioned regions, stratifying BPS by provinces. The sample size was calculated
using the following Equation (1) [45]:

Z2α pq
n= (1)
L2
where n represents the sample size; Zα is the required value for confidence = 1 − α, with α
corresponding to the confidence level; Zα is the percentile of a standard normal distribution
(1 − α/2); p is the expected prevalence of the pathogen; q is (1 − p); and L is the precision
of the estimate or margin of error. Assuming a lack of knowledge about the prevalence of
STEC in BPS in central Chile, the sample size was calculated assuming a prevalence of 50%,
ensuring the highest possible minimum sample size [45]. A confidence level of 95% and a
precision of 5% were also set.
Sampled BPS were selected considering the information obtained from the agricultural
census conducted by Instituto Nacional de Estadística [46] regarding BPS distribution. A
sample size of 84 and 73 BPS was determined for the Metropolitana and Valparaíso regions,
respectively, adding up to a total of 157 BPS.
Additionally, the number of samples to be collected within each BPS was calculated
following Equation (2) [45]:
 (D − 1)
 
1
n = 1 − αD N− (2)
2
Animals 2023, 13, 2444 4 of 15

where n is the sample size; N is the population size; D is the estimated minimum number of
diseased animals in the group; and α = 1 − the confidence level. Considering the detection
of at least 30% of positive animals and that N equals the number of animals by which BPS
are defined, a minimum sample size of 8 animals per BPS was calculated.

2.2. Sample Collection and Microbiological Analysis

Samples were collected directly from the cloaca or rectum of sampled animals using
sterile swabs with Cary-Blair transport medium (Copan® , Brescia, Italy), which were then
labeled with the animal species and assigned a code to each BPS. In cases where it was
needed, environmental feces samples were collected to achieve the required intra-BPS
sample size. These were correspondingly labeled to differentiate them.
Samples were transported and stored at 4 ◦ C at the Centralized Laboratory for
Veterinary Research (LaCIV) of the Faculty of Veterinary and Livestock Sciences of the
University of Chile until further processing. Sample processing protocols, microbiolog-
ical analyses, and confirmatory PCR were carried out following protocols previously
described [16,31,37,47–51].

2.3. Determination of S. enterica and/or STEC Positivity Rate

The positivity rate for S. enterica, STEC, and S. enterica/STEC (including all BPS that
were positive for either of these pathogens) was determined, considering each BPS as an
epidemiological unit. In this manner, positivity rates were calculated at both regional and
provincial levels using the following Equation (3) [45,52]:

Positive cases
P= (3)
Population at risk

where positive cases correspond to the total number of agent-positive BPS (be it for only
one agent or both, depending on the case) and the population at risk encompasses the total
number of BPS sampled in a given geopolitical unit (region or province).
Additionally, choropleth maps representing BPS positivity rates at province level were
constructed for each pathogen, applying a graded color scale based on calculated ranges
for BPS positivity rates. Choropleth maps were generated using QGis [53].

2.4. Determination of Risk Factors for S. enterica and/or STEC Positivity

A previously validated questionnaire was applied to each BPS [16] to characterize
animal management, biosecurity measures applied, and socio-demographic variables
affecting each BPS. All BPS managers who agreed to participate did so after signing an
informed consent form, complying with the protocols of bioethics and responsibility in
scientific research and biosafety.
Three multivariable logistic regression models were constructed in order to evaluate
the relationship between potential explanatory variables and BPS positivity for either
S. enterica, STEC, or both of them (S. enterica/STEC model) [45]. This last model was
included because, as previously stated, they share common epidemiological characteristics.
In these models, the response variable (Y) is dichotomous, because it can only take two
values, where Y = 0 and Y = 1 represent the absence and presence of one or both pathogens
in each BPS, respectively.
All variables were subjected to a simple logistic regression, selecting those with a
liberal p-value of equal to or less than 0.15 for each model. All those that met this criterion
were further analyzed using Spearman’s correlation (quantitative variables) and Fisher’s
exact test (qualitative variables) to check for collinearity and association between variables,
allowing for correction of potential confounding factors.
Subsequently, multivariable models were built using a stepwise backward elimination
procedure, removing from the model those variables that, when taken out of the models,
did not cause a significant change in its likelihood. This was evaluated using a likelihood
ratio test [54], eliminating variables that gave p-values higher than 0.05 after being removed
Animals 2023, 13, 2444 5 of 15

from the model. Other criteria used to evaluate the elimination of a variable were associated
with the variation caused by the removal of a variable over the coefficients of the rest of
the variables, retaining variables that, after being eliminated, caused a change in other
variables’ regression coefficients of 20% or more. The convergence of the models was
set to a value of epsilon (ε) = e−16 to guarantee an adequate level of stringency for the
models performed. The goodness of fit of the model to the data was assessed using the
Hosmer–Lemeshow test [54,55].
All analyses were performed using R statistical software version 4.2.2 [56] and RStudio
version 2022.12.0+353 [57], using the “nlme” [58], “lme4” [59], “car” [60], “ggplot2” [61],
and “ResourceSelection” [62] packages.

2.5. Determination of Antimicrobial Resistance in S. enterica and STEC Isolates

All S. enterica and STEC isolates for which recovery was achieved were evaluated
by the minimum inhibitory concentration (MIC) method to determine antimicrobial re-
sistance profiles. In order to calculate MIC, an automated VITEK® 2 system (bioMérieux,
Marcy-l’Etoile, France), calibrated with reference strains was used according to manufac-
turer’s instructions, employing AST-GN98 cards for the evaluation. The antimicrobial
panel evaluated consisted of drugs commonly used in veterinary and human medicine,
including: aminoglycosides (amikacin and gentamicin); β-lactams (amoxicillin-clavulanic
acid, ampicillin, cephalexin, cefovecin, cefpodoxime, ceftazidime, ceftiofur, and imipenem);
folate synthesis inhibitors (trimethoprim-sulfamethoxazole); nitrofurans (nitrofurantoin);
phenicols (chloramphenicol); quinolones (ciprofloxacin, enrofloxacin, and marbofloxacin);
tetracyclines (doxycycline); and also determines the presence or absence of the extended
spectrum β-lactamase (ESBL) phenotype of the pathogen under study, using a combination
of cefepime, cefotaxime, and ceftazidime alone and in combination with clavulanic acid.
Clinical cut-off values were applied according to the Clinical Laboratory Standards
Institute [63] and the European Committee on Antimicrobial Susceptibility Testing [64],
considering strains with intermediate susceptibility as resistant. Strains resistant to three or
more antimicrobial classes were considered multidrug resistant (MDR) [65].

3. Results
3.1. S. enterica and STEC Positivity Rate in BPS from Valparaíso and Metropolitana Regions
A total of 139 (89%; 139/157) BPS were sampled, achieving the required number of
sampled BPS in the Metropolitana region but only 74% (54/73) of the required samples for
the Valparaíso region. This was due to health and movement restrictions associated with
the COVID-19 pandemic, managing to collect 44.44% (8/18) of the samples required from
the Petorca province and none (0/11) from the Quillota province (Table 1).
The geographical area of study (Metropolitana and Valparaíso regions) showed a
positivity rate of 2.88% (4/139) at the BPS level for S. enterica. This pathogen was found
only in the Metropolitana region, with a positivity rate of 4.71% (4/85) at the regional level.
In this region, cases were majorly concentrated in the provinces of Melipilla, Cordillera,
and Maipo. Animal species associated with S. enterica-positive samples included chickens
and a goose. The positivity rate of STEC at the BPS level determined for both regions was
14.39% (20/139). The Metropolitana region presented a positivity rate of 11.76% (10/85),
with STEC-positive BPS found in four of the six provinces contained within this region,
corresponding to: Melipilla, Cordillera, Maipo, and Chacabuco. On the other hand, the
Valparaíso region presented a positivity rate of 18.52% (10/54), with positive BPS found in
three of the six provinces studied, corresponding to: San Felipe, San Antonio, and Petorca.
STEC-positive animal species included pigs, ducks, chickens, geese, cows, sheep, and goats.
When evaluating Enterobacteriaceae, the positivity rate for the studied geographical area
was 17.27% (24/139). In the Metropolitan region, a positivity rate of 16.47% (14/85) was
found, determining the presence of the pathogens in the provinces of Melipilla, Cordillera,
Maipo, and Chacabuco. Positivity rate values for Enterobacteriaceae in the Valparaíso region
 Los Andes 3 0 0% 0 0% 0 0%
de Valpara-
6 0 0% 0 0% 0 0%
íso
San Felipe 25 0 0% 6 24% 6 24%
Animals 2023, 13, 2444 6 of 15
Valparaíso San Anto-
12 0 0% 3 25% 3 25%
nio
Petorca 8 0 0% 1 12.50% 1 12.50%
Quillota 0 0 0%
were the same as for STEC, since 0 0%the
none of sampled BPS 0
in this region were 0%
positive for
Subtotal 54 0 0%
S. enterica (Figure 1; Table 1). 10 18.52% 10 18.52%
Total 139 4 2.88% 20 14.39% 24 17.27%
Table 1. Backyard production systems sampled and positivity rate by province and region.
The geographical area of study (Metropolitana and Valparaíso regions) showed a
◦ ◦ of S.
positivity rateNofof2.88% (4/139)
Positivity at the BPS N◦level
of for S. enterica. ThisN pathogen
Positivity
Positivity
was found
N◦ of BPS S. enterica- Rate of enterica/ Rate of
Region Province only in the Metropolitana STEC-Positive Rate of
Sampled Positive S.region,
entericawith a positivity rate of 4.71% (4/85) at the regional
STEC-Positive level.
S. enterica/
BPS STEC (%)
In this region,BPScases were majorly
(%) concentrated in the provinces of BPS Melipilla, Cordillera,
STEC (%)
Melipilla and34 Maipo. Animal
2 species5.88%
associated with4S. enterica-positive
11.77% samples 6 included chickens
17.65%
Talagante and 7 a goose. The0 positivity rate 0% of STEC at 0the BPS level0% determined for 0 both regions 0% was
Cordillera 5 1 20% 3 60% 4 80%
Maipo 14.39%
16 (20/139).1 The Metropolitana
6.25% region1 presented 6.25%
a positivity rate 2 of 11.76%12.50%
(10/85),
Metropolitana
Chacabuco with
13 STEC-positive
0 BPS found 0% in four of the 2 six provinces
15.38% contained2 within this15.38%region,
Santiago 10
corresponding 0to: Melipilla,0% Cordillera, Maipo,0 0%
and Chacabuco. On 0the other hand, 0% the
Subtotal 85
Valparaíso 4 presented
region 4.71%
a positivity rate10 of 18.52%11.76% 14
(10/54), with positive 16.47% in
BPS found
Los Andes three
3 of the six provinces
0 studied,
0% corresponding
0 to: San0%
Felipe, San Antonio,
0 and Petorca.
0%
de
STEC-positive
6 0animal species 0% included pigs, 0 ducks, chickens,
0% geese,0 cows, sheep, 0% and
Valparaíso
San Felipe goats.
25 When evaluating
0 Enterobacteriaceae,
0% the
6 positivity rate for the studied
24% 6 geographical
24%
Valparaíso
San area
12 was 17.27% 0 (24/139). In0% the Metropolitan 3 region, 25%
a positivity rate3 of 16.47% 25% (14/85)
Antonio
Petorca
was 8
found, determining
0
the presence
0%
of the1
pathogens in
12.50%
the provinces1
of Melipilla,
12.50%
Cor-
Quillota dillera,
0 Maipo, 0and Chacabuco. 0% Positivity rate
0 values for
0% Enterobacteriaceae
0 in the Valpa-
0%
Subtotal raíso
54 region were 0 the same as 0%for STEC, since 10 none of the sampled BPS
18.52% 10 in this region
18.52%were
Total
positive
139
for S. enterica
4
(Figure
2.88%
1; Table 1). 20 14.39% 24 17.27%

Figure 1. Choropletic maps of S. enterica, STEC, and S. enterica/STEC positivity rates by province in
Figure 1. Choropletic
the Metropolitana maps
and of S. enterica,
Valparaíso regionsSTEC, and S.
of Chile. enterica/STEC
(A) positivityinrates
Provinces evaluated this by province
study in
and their
the Metropolitana
respective region.and Valparaíso
(B–D) shows S.regions
enterica,ofSTEC,
Chile.and
(A) S.
Provinces evaluated
enterica/STEC in thisrates
positivity study
byand their
province,
respective region. (B–D) shows S. enterica, STEC, and S. enterica/STEC positivity rates by province,
respectively.
respectively.
3.2. Risk Factor Analysis for S. enterica, STEC, and S. enterica/STEC in BPS from Valparaíso and
Metropolitana Regions
The multivariate logistic regression model selected for S. enterica is shown in Table 2;
the results of the univariable logistic regression analysis can be observed in detail in
Supplementary Table S1. In this model, only the contact between BPS animals and wild
birds was determined as a factor that decreases the risk of positivity to S. enterica (odds
ratio (OR) = 0.06; 95% confidence interval (95% CI) = 0.00–0.064; p = 0.02).
Animals 2023, 13, 2444 7 of 15

Table 2. Logistic regression model associated with risk factor determination for S. enterica positivity
in BPS.

95% CI 2
Variable Category p-Value OR 1 Lower Upper
(Intercept) - 0.352 0.486 0.106 2.221
N◦ of pets - 0.160 0.541 0.229 1.277
No reference
Animals have contact with wild birds
Yes 0.019 0.059 0.005 0.636
1 Odds ratio; 2 confidence interval.

The multivariate logistic regression model selected for STEC is shown in Table 3. In
this model, only the presence of ruminants inside the BPS (OR = 1.03; 95% CI = 1.00–1.07;
p = 0.036) was identified as a factor that raises the risk of positivity to STEC.

Table 3. Logistic regression model associated with risk factor determination for Shiga toxin-producing
Escherichia coli positivity in BPS.

95% CI 2
Variable Category p-Value OR 1 Lower Upper
(Intercept) - <0.001 0.027 0.003 0.218
Presence of ruminants - 0.036 1.038 1.002 1.075
Presence of guinea pigs or rabbits - 0.132 1.126 0.964 1.316
No reference
Animals have contact with wild birds
Yes 0.251 3.429 0.417 28.179
No reference
Receive state assistance or support
Yes 0.266 1.901 0.612 5.894
1 2
Odds ratio; confidence interval.

The model of multivariate logistic regression selected for S. enterica/STEC is shown

in Table 4. In this model, two variables were identified as factors that alter the risk for
S. enterica/STEC positivity in BPS, both increasing it. These variables correspond to the
presence of ruminants inside BPS (OR = 1.05; 95% CI = 1.02–1.09; p = 0.004) and animal
handling being exclusively executed by women (OR = 3.54; 95% CI = 1.03–12.19; p = 0.045).

Table 4. Logistic regression model associated with risk factor determination for S. enterica/STEC
positivity in BPS.

95% CI 2
Variable Category p-Value OR 1 Lower Upper
(Intercept) - <0.001 0.027 0.003 0.218
Presence of ruminants - 0.004 1.038 1.002 1.075
Family reference
Person in charge of the system Man 0.213 2.422 0.603 9.732
Woman 0.045 3.542 1.029 12.193
1 Odds ratio; 2 confidence interval.

3.3. Antimicrobial Resistance Profiles of S. enterica and STEC Isolates

A total of 5/5 S. enterica and 14/37 STEC strains were recovered, and minimum in-
hibitory concentration (MIC) analysis was performed on all of them (Supplementary Table S2).
S. enterica isolates came from two BPS located in Melipilla, one BPS located in Maipo and
one BPS located in Cordillera; all provinces belonging to the Metropolitana region. On
the other hand, STEC isolates came from three BPS located in Melipilla, two BPS located
in Chacabuco and one BPS located in Cordillera. The antimicrobial resistance profiles
obtained from MIC analysis can be seen in Table 5.
Animals 2023, 13, 2444 8 of 15

Table 5. Antimicrobial resistance profiles identified in S. enterica and STEC isolates, according to MIC
results.

N◦ of Antimicrobial
Pathogen (N◦ of Antimicrobial
N◦ of Isolates Frequency Drugs with Strain
Isolates) Resistance Profile
Resistance
1 20% CLX, IMP, DXC, NTF * 4
1 20% CLX, CVN, CPN 3
1 20% AMP, AMX, DXC, NTF, CPN 5
S. enterica (5)
AMP, AMX, CTD, DXC, NTD,
1 20% 6
CPN
1 20% - 0
12 85.7% CLX 1
STEC (14)
2 14.3% CLX, CPN 2
* CLX: Cephalexin, IMP: Imipenem, DXC: Doxycycline, NTF: Nitrofurantoin (NTF), CVN: Cefovecin, CPN:
Chloramphenicol, AMP: Ampicillin, AMX: Amoxicillin with clavulanic acid, and CTD: Ceftazidime.

Five antimicrobial resistance profiles were identified in S. enterica isolates. Each of them
with a frequency of 20% (1/5). Four of these profiles were identified as MDR, presenting
resistance to Betalactams, Tetracyclines, Nitrofurans, and Amphenicols, and one was found
to be fully antimicrobial-sensitive. In the case of STEC isolates, two antimicrobial resistance
profiles were identified. One had a frequency of 85.7% (12/14) and the other profile showed
a frequency of 14.3% (2/14). None of the STEC isolates were found to be MDR.

4. Discussion
Salmonella enterica and STEC are zoonotic pathogens that can cause potentially severe
or lethal disease in at-risk populations. Notably, BPS are important drivers of disease
transmission and maintenance for both pathogens. Occasionally, both animal handlers
and family members present in these systems may be considered at-risk populations, due
to their age range, reproductive status, or presence of comorbidities leading to immuno-
suppression. These family groups may be exposed to these pathogens due to direct and
indirect contact with their animals, feces, and the products they obtain from them.
Poultry is the most frequently reported reservoir for S. enterica [15,16,22]. This is
consistent with the results obtained in this study, since positivity for S. enterica was only ob-
served in poultry. On the other hand, STEC has been described in several reservoir species
which could be present in BPS, with ruminants acting as the main reservoirs [12–14,66].
In the same manner, this study identified ruminants as the main reservoirs, followed by
poultry and pigs.
For the whole area under study, S. enterica positivity rate at BPS level was 2.88%,
a result similar to the 2.9% reported in San Lorenzo, Paraguay in productions keeping
poultry of different stages [67], a situation comparable to BPS in Chile keeping animals of
various origins, breeds, and stages. In contrast, this value is higher than that of Entre Rios,
Argentina [68], and lower than other reports made around the world, from West Bengal,
India [69]; South Australia [70]; northwestern Nigeria [71]; central Ecuador [72]; and Tien Gi-
ang, Vietnam [73]. This implies that the health status of S. enterica in Chile is good when com-
pared to most countries, which could be explained by variables not analyzed in this study,
related to climatic conditions, interaction networks and/or host–pathogen interactions.
In Chile, previously reported data for S. enterica positivity rate in the Metropolitana
region was 8.3%, while 6.6% was recorded in the Valparaíso region [16]. In contrast, the
regional positivity rates determined in this study were lower than those reported previously
for both studied regions. At the province level, in the Metropolitan region, the province
of Melipilla presented a lower positivity rate than previously reported [16]. Meanwhile,
the provinces of Maipo and Cordillera presented values of 6.25% and 20%, respectively;
findings not previously described. Contrary to this, the province of Chacabuco in the
Metropolitana region, and the provinces of San Antonio (5% and 20%) and San Felipe de
Aconcagua (10%) in the Valparaíso region, were negative for S. enterica in this study, when
Animals 2023, 13, 2444 9 of 15

previous studies have detected circulation for this pathogen in them [16,74]. It is important
to mention that, since a cross-sectional study was carried out, positivity rates values could
have been underestimated at both the regional and province level. Therefore, given the
low positivity rate reported for S. enterica in BPS, it is suggested that future studies reduce
the study area in order to increase the number of samples per province or to perform
longitudinal epidemiological studies.
In Chile, there are reports of STEC in slaughterhouses, zoos, and livestock
farms [39,75,76], but information on BPS animals is scarce. A higher positivity rate for
S. enterica was expected, considering that poultry were the most frequently raised species in
these systems; nevertheless, a STEC positivity rate five times higher than that of S. enterica
was found, highlighting the importance of investing in more research on this pathogen and
its circulation in BPS animals. This finding emphasizes the need to educate people about
the latent risks of zoonoses associated with BPS animals, given that clinical cases of STEC
in the United States and Chile were associated with contact with infected animals [23,77].
Regarding risk factors, this study suggests that potential contact between BPS animals
and wild birds is a factor that reduces the risk of S. enterica positivity, although biosecurity
measures for commercial farms indicate the opposite [28]. Previous studies in wild bird
populations from Chile have found a low prevalence of S. enterica [78], although their
significance for Enterobacteriaceae epidemiology has not been previously addressed in
this country. This result could be an indirect indicator of the available surface to the
animals for their movement, where they would potentially have contact with wild birds.
A larger surface area for movement could imply a greater dispersal of feces, decreasing
the probability of contact of a susceptible animal with feces from an infected animal; thus,
reducing the infection pressure of the pathogen. Further studies should address the role
that wildlife animals play in Enterobacteriaceae transmission in BPS. On the other hand, the
rearing of ruminant species in BPS was identified as a factor that increases the risk of STEC
positivity, an expected result given the high prevalence of STEC described in ruminants [79].
This highlights the importance of identifying, characterizing, and monitoring this pathogen
in BPS animal species.
In the S. enterica/STEC model, the presence of ruminant species in BPS and the animal
handler being a woman increased the risk of positivity. This last point is relevant because
the majority of BPS managers are women [37,42], and a large percentage are pensioners
(over 60 years old) [31]. In Chile, it is common to find women dedicated mostly to domestic
labor and caring for people, especially in rural areas [80].
The dedication of time to caring for others and to domestic chores leads to a reduction
in the time spent handling animals. Moreover, it is estimated that the average number
of hours worked by rural women was 13 h/day, both inside and outside the home [81].
Additionally, in the forestry and livestock sector, it has been found that women have lesser
access to training [82–84]. Therefore, the lack of time, due to a work overload, and training
for rural women could be limiting their capabilities to manage BPS when compared to
men, leaving time for only basic management, such as feeding and releasing/enclosing
animals and resulting in reduced or absent biosecurity and hygiene measures. Further
data supports gender inequality in rural areas; i.e., the percentage of illiteracy in rural
women (70+ years of age) of Chile was over 35% and labor force participation rate for
rural women was of 19%, 48 percentage points less than rural men and 19 percentage
points less than urban women [83,84]. Currently, in the forestry and livestock sector, the
percentage of employed women corresponds to 24.2% of the total number of workers,
almost 52 percentage points below that of rural men [81]. In this sense, the results of the
present study show an example of a potential threat to public health generated from gender
inequality in a rural context. Based on the above, it is necessary to increase access and to
focus training and education for rural women in productive and biosafety areas, expanding
the real scope of the targeted government programs.
In this study, five strains of S. enterica were isolated, with five different antimicrobial
resistance profiles, 80% of them being MDR. Among the groups of antimicrobials to which
Animals 2023, 13, 2444 10 of 15

resistance was reported were Betalactams, Tetracyclines, Nitrofurans, and Phenicols; similar
results were reported for different animal systems from central Chile [43], which includes
S. enterica isolates from BPS, also observed in isolates from industrial pigs and chickens
in Chile [85]. Within the Betalactams, resistance was reported against Penicillin, first-
and third-generation Cephalosporins, and Carbapenemics, all of which are considered
critically important in human medicine based on five prioritization criteria according to
the World Health Organization (WHO) [86]. In the case of STEC, two AMR profiles were
identified for the 14 isolates; none corresponded to MDR, but all showed resistance to
Cephalexin, and 14.3% to Chloramphenicol, similar to prior reports from industrial animals
in Chile [47]. The resistances reported for STEC in this study are not part of the WHO
category of critically important AM for human medicine [86]. The use of AM in STEC
infections in humans is considered contraindicated due to their potential to increase the
risk of HUS. Antibiotics can eliminate the beneficial intestinal microbiota that competes
with STEC and also cause lysis of the bacterial cell well, leading to an elevated release of
preformed Stx toxins. Moreover, using AM can induce phage production and stx gene
expression, further exacerbating the infection [87]. This is particularly significant in rural
settings where non-prescribed use is often sought as an alternative [31].
Multidrug-resistant S. enterica and STEC isolates have been linked to human out-
breaks worldwide; most of them have an animal [88–92], vegetal [93–95], or environmental
origin [90], causing potential severe clinical outbreak in humans [96], and are detected
frequently in developing and undeveloped countries [97]. Evidence in Chile is scarce,
but food matrices and water sources have been reported to be positive to MDR enteric
pathogens [5,98–100]. In addition, the identification of MDR strains is relevant because of
the therapeutic limitations it could generate in severe bacterial infections and the possibility
of transmission of resistance genes through mobile genetic elements to previously sensitive
bacteria of the same genus or others [101]. The diversity of resistance profiles could be
attributed to the multiple sources of replacement animals, food, and water, and the use of
antimicrobials without veterinary prescription in animals raised in BPS.
Both BPS human and animal populations are neglected. This highlights the need to
reduce the gaps in knowledge of the health status of these people so that information is
available to future researchers and authorities; thus, the need to increase resources for
the characterization and surveillance of these human and animal populations with a One
Health approach is emphasized. Furthermore, the transfer of knowledge to the at-risk
population should be addressed, particularly the education of children through schools and
local media, as they are the most active participants in the education of the family group.

5. Conclusions
This study shows the circulation of S. enterica and STEC strains in different animal
species kept in BPS in central Chile. Additionally, it shows the circulation of strains with
different antimicrobial resistance profiles, detecting fully sensitive isolates and others
multiresistant to AM which are considered critically important under the One Health
approach, because of their use in human bacterial disease treatment and their effects on
animal, environmental, and human health.
Furthermore, the existence of factors that modify the risk for BPS positivity for both
pathogens in central Chile was also evidenced, highlighting the need to educate BPS owners,
especially women, and their families, in topics regarding biosecurity and antimicrobial
stewardship, thus adding to the reduction of gender inequalities in rural communities of
Chile and decreasing the probability for the emergence of zoonotic pathogens showing
AMR, which could threaten animal and human public health in Chile.

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/ani13152444/s1, Table S1: Univariable logistic regression
analysis results for S. enterica, STEC, and S. enterica/STEC models; www.mdpi.com/xxx/s2, Table S2:
Positive BPS samples by recovery state, animal species, region, and BPS-ID.
Animals 2023, 13, 2444 11 of 15

Author Contributions: Conceptualization, R.A.-M., G.R.-T., C.U.-E., B.F.-S. and E.P.-M.; methodology,
R.A.-M., C.U.-E., B.F.-S. and E.P.-M.; software, R.A.-M. and C.U.-E.; validation, R.A.-M., C.U.-E.,
B.F.-S. and E.P.-M.; formal analysis, R.A.-M., C.U.-E., B.F.-S. and E.P.-M.; investigation, R.A.-M.,
C.U.-E. and G.R.-T.; resources, R.A.-M.; data curation, R.A.-M. and C.U.-E.; writing—original draft
preparation, R.A.-M., C.U.-E., B.F.-S. and E.P.-M.; writing—review and editing, R.A.-M., G.R.-T.,
M.L.-T. and A.E.R.; visualization, R.A.-M., C.U.-E., B.F.-S. and E.P.-M.; supervision, R.A.-M.; project
administration, R.A.-M., G.R.-T., M.L.-T. and A.E.R.; funding acquisition, R.A.-M., G.R.-T., M.L.-T.
and A.E.R. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Fondo Nacional de Desarrollo Científico y Tecnológico
(FONDECYT), grant number 11180476 and by Banco Interamericano de Desarrollo (BID)-Fondo
Regional de Tecnología Agropecuaria (FONTAGRO), grant number ATN/RF-18136-RG.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, in accordance with the biosafety standards of a level 2 laboratory, according
to the CONICYT Manual of Biosecurity standards (https://www.conicyt.cl/fondecyt/files/2018/0
8/manual-de-normas-de-bioseguridad.pdf accessed on 15 November 2019), and approved by the
Biosecurity Institutional Review Board (FAVET-UCH permit code 131) and bioethics committee of
CICUA-UChile (permit code 18205-VET-UCH on 27 November 2018).
Informed Consent Statement: Informed consent was obtained from all BPS owners involved
in the study.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data are not publicly available because they are part of an ongoing project
not yet published.
Acknowledgments: The authors acknowledge all members of the Zoonotic Agents Epidemiology
Group and members of the Microbiology Laboratory from Facultad de Ciencias Veterinarias y Pecuar-
ias, Universidad de Chile, for their support in sample and data collection and sample processing.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Engering, A.; Hogerwerf, L.; Slingenbergh, J. Pathogen–host–environment interplay and disease emergence. Emerg. Microbes
Infect. 2013, 2, e5. [CrossRef] [PubMed]
2. Graziani, C.; Losasso, C.; Luzzi, I.; Ricci, A.; Scavia, G.; Pasquali, P. Chapter 5—Salmonella. In Foodborne Diseases, 3rd ed.; Dodd,
C.E.R., Aldsworth, T., Stein, R.A., Cliver, D.O., Riemann, H.P., Eds.; Academic Press: Cambridge, MA, USA, 2017; pp. 133–169.
[CrossRef]
3. Smith, J.L.; Fratamico, P.M. Chapter 7—Escherichia coli as a Pathogen∗. In Foodborne Diseases, 3rd ed.; Dodd, C.E.R., Aldsworth, T.,
Stein, R.A., Cliver, D.O., Riemann, H.P., Eds.; Academic Press: Cambridge, MA, USA, 2017; pp. 189–208. [CrossRef]
4. Zhang, H.; Yamamoto, E.; Murphy, J.; Carrillo, C.; Locas, A. Shiga Toxin–Producing Escherichia coli (STEC) and STEC-Associated
Virulence Genes in Raw Ground Pork in Canada. J. Food Prot. 2021, 84, 1956–1964. [CrossRef] [PubMed]
5. Martínez, M.C.; Retamal, P.; Rojas-Aedo, J.F.; Fernández, J.; Fernández, A.; Lapierre, L. Multidrug-Resistant Outbreak-Associated
Salmonella Strains in Irrigation Water from the Metropolitan Region, Chile. Zoonoses Public Health 2017, 64, 299–304. [CrossRef]
[PubMed]
6. Plowright, R.K.; Parrish, C.R.; McCallum, H.; Hudson, P.J.; Ko, A.I.; Graham, A.L.; Lloyd-Smith, J.O. Pathways to zoonotic
spillover. Nat. Rev. Microbiol. 2017, 15, 502–510. [CrossRef]
7. Smith, J.L.; Fratamico, P.M. Emerging and Re-Emerging Foodborne Pathogens. Foodborne Pathog. Dis. 2018, 15, 737–757. [CrossRef]
8. European Food Safety Authority; European Centre for Disease Prevention and Control. The European Union One Health 2018
Zoonoses Report. EFSA J. 2019, 17, e05926. [CrossRef]
9. CDC. Outbreak of Salmonella Infections Linked to Backyard Poultry. 2020. Available online: https://www.cdc.gov/salmonella/
backyardpoultry-05-20/index.html (accessed on 15 April 2023).
10. Wibisono, F.M.; Wibisono, F.J.; Effendi, M.H.; Plumeriastuti, H.; Hidayatullah, A.R.; Hartadi, E.B.; Sofiana, E.D. A review of
salmonellosis on poultry farms: Public health importance. Syst. Rev. Pharm. 2020, 11, 481–486.
11. Scaife, H.R.; Cowan, D.; Finney, J.; Kinghorn-Perry, S.F.; Crook, B. Wild rabbits (Oryctolagus cuniculus) as potential carriers of
verocytotoxin-producing Escherichia coli. Vet. Rec. 2006, 159, 175–178. [CrossRef]
12. Ferens, W.A.; Hovde, C.J. Escherichia coli O157:H7: Animal Reservoir and Sources of Human Infection. Foodborne Pathog. Dis.
2010, 8, 465–487. [CrossRef]
13. Persad Anil, K.; LeJeune Jefrey, T. Animal Reservoirs of Shiga Toxin-Producing Escherichia coli. Microbiol. Spectr. 2014, 2, 2.4.23.
[CrossRef]
Animals 2023, 13, 2444 12 of 15

14. Bryan, A.; Youngster, I.; McAdam, A.J. Shiga Toxin Producing Escherichia coli. Clin. Lab. Med. 2015, 35, 247–272. [CrossRef]
15. Barreto, M.; Castillo-Ruiz, M.; Retamal, P. Salmonella enterica: Una revisión de la trilogía agente, hospedero y ambiente, y su
trascendencia en Chile. Rev. Chil. Infectología 2016, 33, 547–557. [CrossRef]
16. Alegria-Moran, R.; Rivera, D.; Toledo, V.; Moreno-Switt, A.I.; Hamilton-West, C. First detection and characterization of
Salmonella spp. in poultry and swine raised in backyard production systems in central Chile. Epidemiol. Infect. 2017, 145,
3180–3190. [CrossRef]
17. Dias, D.; Caetano, T.; Torres, R.T.; Fonseca, C.; Mendo, S. Shiga toxin-producing Escherichia coli in wild ungulates. Sci. Total
Environ. 2019, 651, 203–209. [CrossRef]
18. Wang, J.; Li, J.; Liu, F.; Cheng, Y.; Su, J. Characterization of Salmonella enterica Isolates from Diseased Poultry in Northern China
between 2014 and 2018. Pathogens 2020, 9, 95. [CrossRef]
19. Hale, C.R.; Scallan, E.; Cronquist, A.B.; Dunn, J.; Smith, K.; Robinson, T.; Lathrop, S.; Tobin-D’Angelo, M.; Clogher, P. Estimates of
Enteric Illness Attributable to Contact With Animals and Their Environments in the United States. Clin. Infect. Dis. 2012, 54,
S472–S479. [CrossRef]
20. Majowicz, S.E.; Scallan, E.; Jones-Bitton, A.; Sargeant, J.M.; Stapleton, J.; Angulo, F.J.; Yeung, D.H.; Kirk, M.D. Global Incidence of
Human Shiga Toxin–Producing Escherichia coli Infections and Deaths: A Systematic Review and Knowledge Synthesis. Foodborne
Pathog. Dis. 2014, 11, 447–455. [CrossRef]
21. Crump John, A.; Sjölund-Karlsson, M.; Gordon Melita, A.; Parry Christopher, M. Epidemiology, Clinical Presentation, Laboratory
Diagnosis, Antimicrobial Resistance, and Antimicrobial Management of Invasive Salmonella Infections. Clin. Microbiol. Rev. 2015,
28, 901–937. [CrossRef]
22. Eng, S.-K.; Pusparajah, P.; Ab Mutalib, N.-S.; Ser, H.-L.; Chan, K.-G.; Lee, L.-H. Salmonella: A review on pathogenesis, epidemiology
and antibiotic resistance. Front. Life Sci. 2015, 8, 284–293. [CrossRef]
23. ISP. Boletín de Vigilancia de Laboratorio de E. coli Productora de Toxina Shiga. Chile, 2010—2016; Gobierno de Chile: Santiago, Chile,
2017; Volume 7.
24. Saeedi, P.; Yazdanparast, M.; Behzadi, E.; Salmanian, A.H.; Mousavi, S.L.; Nazarian, S.; Amani, J. A review on strategies for
decreasing E. coli O157:H7 risk in animals. Microb. Pathog. 2017, 103, 186–195. [CrossRef]
25. Lee, H.; Yoon, Y. Etiological Agents Implicated in Foodborne Illness World Wide. Food Sci. Anim. Resour. 2021, 41, 1–7. [CrossRef]
[PubMed]
26. Torres, J.; Voisier, A.; Berríos, I.; Pitto, N.; Durán Agüero, S. Knowledge and application in hygienic practices in food preparation
and self-report of food poisoning in Chilean homes. Rev. Chil. Infectología 2018, 35, 483–489. [CrossRef] [PubMed]
27. ISP. Boletín de Vigilancia de Laboratorio Salmonella spp. 2014–2018; Gobierno de Chile: Santiago, Chile, 2019; Volume 9.
28. Conan, A.; Goutard, F.L.; Sorn, S.; Vong, S. Biosecurity measures for backyard poultry in developing countries: A systematic
review. BMC Vet. Res. 2012, 8, 240. [CrossRef] [PubMed]
29. Wong, J.T.; de Bruyn, J.; Bagnol, B.; Grieve, H.; Li, M.; Pym, R.; Alders, R.G. Small-scale poultry and food security in resource-poor
settings: A review. Glob. Food Secur. 2017, 15, 43–52. [CrossRef]
30. Di Pillo, F.; Anríquez, G.; Alarcón, P.; Jimenez-Bluhm, P.; Galdames, P.; Nieto, V.; Schultz-Cherry, S.; Hamilton-West, C. Backyard
poultry production in Chile: Animal health management and contribution to food access in an upper middle-income country.
Prev. Vet. Med. 2019, 164, 41–48. [CrossRef]
31. Pavez-Muñoz, E.; González, C.; Fernández-Sanhueza, B.; Sánchez, F.; Escobar, B.; Ramos, R.; Fuenzalida, V.; Galarce, N.;
Arriagada, G.; Neira, V.; et al. Antimicrobial Usage Factors and Resistance Profiles of Shiga Toxin-Producing Escherichia coli in
Backyard Production Systems from Central Chile. Front. Vet. Sci. 2021, 7, 595149. [CrossRef]
32. Fleming, D.A.; Abler, D.G.; Goetz, S.J. Agricultural trade and poverty in Chile: A spatial analysis of product tradability. Agric.
Econ. 2010, 41, 545–553. [CrossRef]
33. Hamilton-West, C.; Rojas, H.; Pinto, J.; Orozco, J.; Hervé-Claude, L.P.; Urcelay, S. Characterization of backyard poultry production
systems and disease risk in the central zone of Chile. Res. Vet. Sci. 2012, 93, 121–124. [CrossRef]
34. Correia-Gomes, C.; Henry, M.K.; Auty, H.K.; Gunn, G.J. Exploring the role of small-scale livestock keepers for national
biosecurity—The pig case. Prev. Vet. Med. 2017, 145, 7–15. [CrossRef]
35. Cantas, L.; Suer, K. Review: The Important Bacterial Zoonoses in “One Health” Concept. Front. Public Health 2014, 2, 144.
[CrossRef]
36. Correia-Gomes, C.; Sparks, N. Exploring the attitudes of backyard poultry keepers to health and biosecurity. Prev. Vet. Med. 2020,
174, 104812. [CrossRef]
37. Pavez-Muñoz, E.; Fernández-Sanhueza, B.; Urzúa-Encina, C.; Galarce, N.; Alegría-Morán, R. Risk Factors for Positivity to Shiga
Toxin-Producing Escherichia coli and Salmonella enterica in Backyard Production Systems Animals from Metropolitana Region,
Chile: A Threat to Public Health? Int. J. Environ. Res. Public Health 2021, 18, 10730. [CrossRef]
38. Fica, C.A.; Alexandre, S.M.; Prat, M.S.; Fernández, R.A.; Fernández, O.J.; Heitmann, G.I. Changes in epidemiological patterns of
salmonellosis in Chile. Since Salmonella typhi to Salmonella enteritidis. Rev. Chil. Infectología 2001, 18, 85–93. [CrossRef]
39. Galarce, N.; Escobar, B.; Sánchez, F.; Paredes-Osses, E.; Alegría-Morán, R.; Borie, C. Virulence Genes, Shiga Toxin Subtypes,
Serogroups, and Clonal Relationship of Shiga Toxin-Producing Escherichia Coli Strains Isolated from Livestock and Companion
Animals. Animals 2019, 9, 733. [CrossRef]
40. McEwen, S.A.; Collignon, P.J. Antimicrobial Resistance: A One Health Perspective. Microbiol. Spectr. 2018, 6, 6.2.10. [CrossRef]
Animals 2023, 13, 2444 13 of 15

41. Aslam, B.; Khurshid, M.; Arshad, M.I.; Muzammil, S.; Rasool, M.; Yasmeen, N.; Shah, T.; Chaudhry, T.H.; Rasool, M.H.;
Shahid, A.; et al. Antibiotic Resistance: One Health One World Outlook. Front. Cell. Infect. Microbiol. 2021, 11, 1153. [CrossRef]
42. Cornejo, J.; Pokrant, E.; Figueroa, F.; Riquelme, R.; Galdames, P.; Di Pillo, F.; Jimenez-Bluhm, P.; Hamilton-West, C. Assessing
Antibiotic Residues in Poultry Eggs from Backyard Production Systems in Chile, First Approach to a Non-Addressed Issue in
Farm Animals. Animals 2020, 10, 1056. [CrossRef]
43. Rivera, D.; Allel, K.; Dueñas, F.; Tardone, R.; Soza, P.; Hamilton-West, C.; Moreno-Switt, A.I. Screening the Presence of Non-
Typhoidal Salmonella in Different Animal Systems and the Assessment of Antimicrobial Resistance. Animals 2021, 11, 1532.
[CrossRef]
44. Instituto Nacional de Estadística. Censo Agropecuario 2021; Gobierno de Chile: Santiago, Chile, 2023.
45. Dohoo, R.; Martin, W.; Stryhn, H. Methods in Epidemiologic Research, 1st ed.; VER Inc.: Charlottetown, PE, Canada, 2012; p. 890.
46. Instituto Nacional de Estadística. National Agricultural Census; Instituto Nacional de Estadística: Santiago, Chile, 2007.
47. Galarce, N.; Sánchez, F.; Fuenzalida, V.; Ramos, R.; Escobar, B.; Lapierre, L.; Paredes-Osses, E.; Arriagada, G.; Alegría-Morán, R.;
Lincopán, N.; et al. Phenotypic and Genotypic Antimicrobial Resistance in Non-O157 Shiga Toxin-Producing Escherichia coli
Isolated From Cattle and Swine in Chile. Front. Vet. Sci. 2020, 7, 367. [CrossRef]
48. Marier, E.A.; Snow, L.C.; Floyd, T.; McLaren, I.M.; Bianchini, J.; Cook, A.J.C.; Davies, R.H. Abattoir based survey of Salmonella in
finishing pigs in the United Kingdom 2006–2007. Prev. Vet. Med. 2014, 117, 542–553. [CrossRef]
49. Malorny, B.; Hoorfar, J.; Bunge, C.; Helmuth, R. Multicenter Validation of the Analytical Accuracy of Salmonella PCR: Towards an
International Standard. Appl. Environ. Microbiol. 2003, 69, 290–296. [CrossRef] [PubMed]
50. Ranieri, M.L.; Shi, C.; Moreno Switt, A.I.; den Bakker, H.C.; Wiedmann, M. Comparison of Typing Methods with a New Procedure
Based on Sequence Characterization for Salmonella Serovar Prediction. J. Clin. Microbiol. 2013, 51, 1786–1797. [CrossRef] [PubMed]
51. Cebula, T.A.; Payne, W.L.; Feng, P. Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like
toxin type by mismatch amplification mutation assay-multiplex PCR. J. Clin. Microbiol. 1995, 33, 248–250. [CrossRef] [PubMed]
52. Thrusfield, M.; Christley, R. Veterinary Epidemiology, 4th ed.; Wiley-Blackwell: Hoboken, NJ, USA, 2018; p. 896.
53. QGIS-Development-Team. QGIS Geographic Information System, Open Source Geospatial Foundation Project. 2023.
54. Hosmer, D.W.; Lemeshow, S.; Rodney, S. Applied Logistic Regression, 3rd ed.; Wiley: Hoboken, NJ, USA, 2013. [CrossRef]
55. Hosmer, D.W.; Hosmer, T.; Le Cessie, S.; Lemeshow, S. A comparison of goodness-of-fit tests for the logistic regression model.
Stat. Med. 1997, 16, 965–980. [CrossRef]
56. R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2023.
57. RStudio Team. RStudio: Integrated Development for R; RStudio, PBC: Boston, MA, USA, 2023.
58. Pinheiro, J.; Bates, D.; DebRoy, S.; Sarkar, D.; R Core Team. nlme: Linear and Nonlinear Mixed Effects Models; 2021. Available online:
http://CRAN.R-project.org/package=nlme (accessed on 15 April 2023).
59. Bates, D.; Mächler, M.; Bolker, B.; Walker, S. Fitting Linear Mixed-Effects Models Using lme4. J. Stat. Softw. 2015, 67, 1–48.
[CrossRef]
60. Fox, J.; Weisberg, S. An R Companion to Applied Regression, 3rd ed.; Sage: Thousand Oaks, CA, USA, 2019.
61. Wickham, H. ggplot2. WIREs Comput. Stat. 2011, 3, 180–185. [CrossRef]
62. Lele, S.; Keim, J.; Solymos, P. ResourceSelection: Resource Selection (Probability) Functions for Use-Availability. Data 2019.
Available online: https://github.com/psolymos/ResourceSelection (accessed on 15 April 2023).
63. Twenty-Fifth Informational Suppl. M100-S25; Performance Standards for Antimicrobial Susceptibility Testing. Clinical and
Laboratory Standars Institute (CLSI): Wayne, PA, USA, 2015.
64. European Committee on Antimicrobial Susceptibility Testing. Clinical Breakpoint Tables for Interpretation of MICs and Zone Diameters;
Version 13.0; European Committee on Antimicrobial Susceptibility Testing: Vaxjó, Sweden, 2023.
65. Magiorakos, A.P.; Srinivasan, A.; Carey, R.B.; Carmeli, Y.; Falagas, M.E.; Giske, C.G.; Harbarth, S.; Hindler, J.F.; Kahlmeter, G.;
Olsson-Liljequist, B.; et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert
proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 2012, 18, 268–281. [CrossRef]
66. Himsworth, C.G.; Zabek, E.; Desruisseau, A.; Parmley, E.J.; Reid-Smith, R.; Jardine, C.M.; Tang, P.; Patrick, D.M. Prevalence and
characteristics of Escherichia coli and Salmonella spp. in the feces of wild urban Norway and black rats (Rattus norvegicus and
Rattus rattus) from an inner-city neighborhood of Vancouver, Canada. J. Wildl. Dis. 2015, 51, 589–600. [CrossRef] [PubMed]
67. Leotta, G.; Suzuki, K.; Alvarez, F.; Nunez, L.; Silva, M.; Castro, L.; Faccioli, M.; Zarate, N.; Weiler, N.; Alvarez, M. Prevalence of
Salmonella spp. in backyard chickens in Paraguay. Int. J. Poult. Sci. 2010, 9, 533–536. [CrossRef]
68. Rodríguez, F.I.; Pascal, D.C.; Pulido, D.; Osinalde, J.M.; Caffer, M.I.; Bueno, D.J. Prevalence, antimicrobial resistance profile and
comparison of selective plating media for the isolation of Salmonella in backyard chickens from Entre Rios, Argentina. Zoonoses
Public Health 2018, 65, e95–e101. [CrossRef]
69. Banerjee, A.; Bardhan, R.; Chowdhury, M.; Joardar, S.N.; Isore, D.P.; Batabyal, K.; Dey, S.; Sar, T.K.; Bandyopadhyay, S.;
Dutta, T.K.; et al. Characterization of beta-lactamase and biofilm producing Enterobacteriaceae isolated from organized and
backyard farm ducks. Lett. Appl. Microbiol. 2019, 69, 110–115. [CrossRef] [PubMed]
70. Manning, J.; Gole, V.; Chousalkar, K. Screening for Salmonella in backyard chickens. Prev. Vet. Med. 2015, 120, 241–245. [CrossRef]
[PubMed]
71. Jibril, A.H.; Okeke, I.N.; Dalsgaard, A.; Kudirkiene, E.; Akinlabi, O.C.; Bello, M.B.; Olsen, J.E. Prevalence and risk factors of
Salmonella in commercial poultry farms in Nigeria. PLoS ONE 2020, 15, e0238190. [CrossRef]
Animals 2023, 13, 2444 14 of 15

72. Salazar, G.A.; Guerrero-López, R.; Lalaleo, L.; Avilés-Esquivel, D.; Vinueza-Burgos, C.; Calero-Cáceres, W. Presence and diversity
of Salmonella isolated from layer farms in central Ecuador. F1000Research 2019, 8, 235. [CrossRef]
73. Trung, N.V.; Carrique-Mas, J.J.; Nghia, N.H.; Tu, L.T.P.; Mai, H.H.; Tuyen, H.T.; Campbell, J.; Nhung, N.T.; Nhung, H.N.;
Minh, P.V.; et al. Non-Typhoidal Salmonella Colonization in Chickens and Humans in the Mekong Delta of Vietnam. Zoonoses
Public Health 2017, 64, 94–99. [CrossRef]
74. Salas Soto, R.A. Salmonella spp., Resistencia a Antimicrobianos y Caracterización de Medidas de Bioseguridad en Sistemas
Productivos de Traspatio Vecinos a La Reserva Nacional El Yali. Master’s Thesis, Universidad de Chile, Santiago, Chile, 2016.
75. Marchant, P.; Hidalgo-Hermoso, E.; Espinoza, K.; Retamal, P. Prevalence of Salmonella enterica and Shiga toxin-producing
Escherichia coli in zoo animals from Chile. J. Vet. Sci. 2016, 17, 583–586. [CrossRef]
76. Díaz, L.; Gutierrez, S.; Moreno-Switt, A.I.; Hervé, L.P.; Hamilton-West, C.; Padola, N.L.; Navarrete, P.; Reyes-Jara, A.; Meng, J.;
González-Escalona, N.; et al. Diversity of Non-O157 Shiga Toxin-Producing Escherichia coli Isolated from Cattle from Central and
Southern Chile. Animals 2021, 11, 2388. [CrossRef]
77. Vachon, M.S.; Khalid, M.; Tarr, G.A.M.; Hedberg, C.; Brown, J.A. Farm animal contact is associated with progression to Hemolytic
uremic syndrome in patients with Shiga toxin-producing Escherichia coli—Indiana, 2012–2018. One Health 2020, 11, 100175.
[CrossRef]
78. Tardone, R.; Rivera, D.; Dueñas, F.; Sallaberry-Pincheira, N.; Hamilton-West, C.; Adell, A.D.; Moreno-Switt, A.I. Salmonella in
Raptors and Aquatic Wild Birds in Chile. J. Wildl. Dis. 2020, 56, 707–712. [CrossRef]
79. Kim, J.-S.; Lee, M.-S.; Kim, J.H. Recent Updates on Outbreaks of Shiga Toxin-Producing Escherichia coli and Its Potential Reservoirs.
Front. Cell. Infect. Microbiol. 2020, 10, 273. [CrossRef]
80. Ballara, M.; Parada, S. El Empleo De Las Mujeres Rurales: Lo Que Dicen Las Cifras; Comisión Economica para América Latina y el
Caribe (CEPAL): Santiago, Chile, 2009.
81. Fundación Promoción y Desarrollo de la Mujer (PRODEMU). Mujeres en la Agricultura Familiaer Campesina en Chile; PRODEMU:
Santiago, Chile, 2021.
82. Fawaz-Yissi, M.J.; Rodriguez-Garces, C. Rural women and work in central Chile. Attitudes, factors and meanings. Cuad. Desarro.
Rural 2013, 70, 47–68.
83. Boza Martínez, S.; Cortés Belmar, M.; Muñoz Eulogio, T. Estrategias de desarrollo rural con enfoque de género en Chile: El caso
del programa “Formación y capacitación para mujeres campesinas”. Civ. Cienc. Soc. Y Hum. 2016, 16, 63–76.
84. Mestmacher, J.; Braun, A. Women, agroecology and the state: New perspectives on scaling-up agroecology based on a field
research in Chile. Agroecol. Sustain. Food Syst. 2021, 45, 981–1006. [CrossRef]
85. Retamal, P.; Gaspar, J.; Benavides, M.B.; Saenz, L.; Galarce, N.; Aravena, T.; Cornejo, J.; Lapierre, L. Virulence and antimicrobial
resistance factors in Salmonella enterica serotypes isolated from pigs and chickens in central Chile. Front. Vet. Sci. 2022, 9, 971246.
[CrossRef]
86. World Health Organization (WHO). Critically Important Antimicrobials for Human Medicine; World Health Organization: Geneva,
Switzerland, 2019.
87. Kakoullis, L.; Papachristodoulou, E.; Chra, P.; Panos, G. Shiga toxin-induced haemolytic uraemic syndrome and the role of
antibiotics: A global overview. J. Infect. 2019, 79, 75–94. [CrossRef]
88. Rueda-Furlan, J.P.; Gallo, I.F.L.; de Campos, A.C.L.P.; Passaglia, J.; Falcão, J.P.; Navarro, A.; Nakazato, G.; Stehling, E.G. Molecular
characterization of multidrug-resistant Shiga toxin-producing Escherichia coli harboring antimicrobial resistance genes obtained
from a farmhouse. Pathog. Glob. Health 2019, 113, 268–274. [CrossRef]
89. Furlan, J.P.R.; Ramos, M.S.; dos Santos, L.D.R.; da Silva Rosa, R.; Stehling, E.G. Multidrug-resistant Shiga toxin-producing
Escherichia coli and hybrid pathogenic strains of bovine origin. Vet. Res. Commun. 2023. [CrossRef]
90. Lalhruaipuii, K.; Dutta, T.K.; Roychoudhury, P.; Chakraborty, S.; Subudhi, P.K.; Samanta, I.; Bandyopadhayay, S.; Singh, S.B.
Multidrug-Resistant Extended-Spectrum β-Lactamase-Producing Escherichia coli Pathotypes in North Eastern Region of India:
Backyard Small Ruminants–Human–Water Interface. Microb. Drug Resist. 2021, 27, 1664–1671. [CrossRef]
91. Pan, Y.; Hu, B.; Bai, X.; Yang, X.; Cao, L.; Liu, Q.; Sun, H.; Li, J.; Zhang, J.; Jin, D.; et al. Antimicrobial Resistance of Non-O157
Shiga Toxin-Producing Escherichia coli Isolated from Humans and Domestic Animals. Antibiotics 2021, 10, 74. [CrossRef]
92. Isler, M.; Wissmann, R.; Morach, M.; Zurfluh, K.; Stephan, R.; Nüesch-Inderbinen, M. Animal petting zoos as sources of Shiga
toxin-producing Escherichia coli, Salmonella and extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. Zoonoses
Public Health 2021, 68, 79–87. [CrossRef]
93. Priyanka; Meena, P.R.; Meghwanshi, K.K.; Rana, A.; Singh, A.P. Leafy greens as a potential source of multidrug-resistant
diarrhoeagenic Escherichia coli and Salmonella. Microbiology 2021, 167, 001059. [CrossRef] [PubMed]
94. Verma, P.; Saharan, V.V.; Nimesh, S.; Singh, A.P. Phenotypic and virulence traits of Escherichia coli and Salmonella strains isolated
from vegetables and fruits from India. J. Appl. Microbiol. 2018, 125, 270–281. [CrossRef] [PubMed]
95. Bautista-De León, H.; Gómez-Aldapa, C.A.; Rangel-Vargas, E.; Vázquez-Barrios, E.; Castro-Rosas, J. Frequency of indicator
bacteria, Salmonella and diarrhoeagenic Escherichia coli pathotypes on ready-to-eat cooked vegetable salads from Mexican
restaurants. Lett. Appl. Microbiol. 2013, 56, 414–420. [CrossRef] [PubMed]
96. Bruyand, M.; Mariani-Kurkdjian, P.; Gouali, M.; de Valk, H.; King, L.A.; Le Hello, S.; Bonacorsi, S.; Loirat, C. Hemolytic uremic
syndrome due to Shiga toxin-producing Escherichia coli infection. Médecine Mal. Infect. 2018, 48, 167–174. [CrossRef]
Animals 2023, 13, 2444 15 of 15

97. Karama, M.; Cenci-Goga, B.T.; Malahlela, M.; Smith, A.M.; Keddy, K.H.; El-Ashram, S.; Kabiru, L.M.; Kalake, A. Virulence
Characteristics and Antimicrobial Resistance Profiles of Shiga Toxin-Producing Escherichia coli Isolates from Humans in South
Africa: 2006–2013. Toxins 2019, 11, 424. [CrossRef]
98. Sánchez, F.; Fuenzalida, V.; Ramos, R.; Escobar, B.; Neira, V.; Borie, C.; Lapierre, L.; López, P.; Venegas, L.; Dettleff, P.; et al.
Genomic features and antimicrobial resistance patterns of Shiga toxin-producing Escherichia coli strains isolated from food in
Chile. Zoonoses Public Health 2021, 68, 226–238. [CrossRef]
99. Aravena, C.; Valencia, B.; Villegas, A.; Ortega, M.; Fernández, R.A.; Araya, R.P.; Saavedra, A.; Del Campo, R. Characterization of
Salmonella Heidelberg strains isolated in Chile. Rev. Médica Chile 2019, 147, 24–33. [CrossRef]
100. Pardo-Esté, C.; Lorca, D.; Castro-Severyn, J.; Krüger, G.; Alvarez-Thon, L.; Zepeda, P.; Sulbaran-Bracho, Y.; Hidalgo, A.; Tello,
M.; Molina, F.; et al. Genetic Characterization of Salmonella Infantis with Multiple Drug Resistance Profiles Isolated from a
Poultry-Farm in Chile. Microorganisms 2021, 9, 2370. [CrossRef]
101. Baquero, F.; Coque, T.M.; Martínez, J.-L.; Aracil-Gisbert, S.; Lanza, V.F. Gene Transmission in the One Health Microbiosphere and
the Channels of Antimicrobial Resistance. Front. Microbiol. 2019, 10, 2892. [CrossRef]

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