antibiotics

Article
Antimicrobial Resistance of Escherichia coli Isolates
from Livestock and the Environment in Extensive Smallholder
Livestock Production Systems in Ethiopia
Biruk Alemu Gemeda 1,2, * , Barbara Wieland 3,4 , Gezahegn Alemayehu 1 , Theodore J. D. Knight-Jones 1 ,
Hiwot Desta Wodajo 1 , Misgana Tefera 2 , Adem Kumbe 5 , Abebe Olani 6 , Shubisa Abera 6 and Kebede Amenu 1,2

1 Animal and Human Health Research Program, International Livestock Research Institute (ILRI),
Addis Ababa P.O. Box 5689, Ethiopia; [email protected] (G.A.);
[email protected] (T.J.D.K.-J.); [email protected] (H.D.W.); [email protected] (K.A.)
2 College of Veterinary Medicine and Agriculture, Addis Ababa University, Bishoftu P.O. Box 1176, Ethiopia;
[email protected]
3 Institute of Virology and Immunology, 3147 Mittelhaeusern, Switzerland; [email protected]
4 Department of Infectious Diseases and Pathobiology (DIP), Vetsuisse Faculty, University of Bern,
3012 Bern, Switzerland
5 Oromia Agricultural Research Institute, Yabello Pastoral and Dryland Agriculture Research Center,
Yabello P.O. Box 85, Ethiopia; [email protected]
6 Animal Health Institute (AHI), Sebeta P.O. Box 04, Ethiopia; [email protected] (A.O.);
[email protected] (S.A.)
* Correspondence: [email protected]

Abstract: The objective of this study was to characterize the distribution of antimicrobial resistance
(AMR) of Escherichia coli (E. coli) isolated from livestock feces and soil in smallholder livestock systems.
Citation: Gemeda, B.A.; Wieland, B.;
A cross-sectional study was carried out sampling 77 randomly selected households in four districts
Alemayehu, G.; Knight-Jones, T.J.D.; representing two agroecologies and production systems. E. coli was isolated and the susceptibility to
Wodajo, H.D.; Tefera, M.; Kumbe, A.; 15 antimicrobials was assessed. Of 462 E. coli isolates tested, resistance to at least one antimicrobial
Olani, A.; Abera, S.; Amenu, K. was detected in 52% (43.7–60.8) of isolates from cattle fecal samples, 34% (95% CI, 26.2–41.8) from
Antimicrobial Resistance of sheep samples, 58% (95% CI, 47.9–68.2) from goat samples and 53% (95% CI, 43.2–62.4) from soil
Escherichia coli Isolates from Livestock samples. AMR patterns for E. coli from livestock and soil showed some similarities, with the highest
and the Environment in Extensive prevalence of resistance detected against streptomycin (33%), followed by amoxycillin/clavulanate
Smallholder Livestock Production (23%) and tetracycline (8%). The odds of detecting E. coli resistance to ≥2 antimicrobials in livestock
Systems in Ethiopia. Antibiotics 2023,
fecal samples were nearly three times (Odd Ratio—OR: 2.9; 95% CI, 1.72–5.17; p = 0.000) higher in
12, 941. https://doi.org/10.3390/
lowland pastoral than in highland mixed crop–livestock production systems. These findings provide
antibiotics12050941
insights into the status of resistance in livestock and soil, and associated risk factors in low-resource
Academic Editors: Adrian settings in Ethiopia.
Zaragoza-Bastida and Nallely
Rivero-Perez Keywords: antimicrobial resistance; livestock; soil; E. coli; smallholders
Received: 14 April 2023
Revised: 16 May 2023
Accepted: 19 May 2023
Published: 22 May 2023 1. Introduction
Antimicrobial resistance (AMR) has been recognized as one of the most significant
threats to the health of people and food-producing animals. The report from the World
Health Organization (WHO) on AMR indicates that resistance of common bacteria has
Copyright: © 2023 by the authors.
reached alarming levels in many parts of the world. For example, the resistance of
Licensee MDPI, Basel, Switzerland.
Escherichia coli (E. coli) and Klebsiella spp. to last-resort third-generation cephalosporins
This article is an open access article
and carbapenems antibiotics has reached up to 54% [1,2]. Some reports estimated that the
distributed under the terms and
economic loss due to AMR will increase dramatically, causing trillion-dollar losses by the
conditions of the Creative Commons
mid-21st century [3]. In line with this, the 2030 Agenda for Sustainable Development Goals
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
emphasized the need to address growing antimicrobial resistance [4,5].
4.0/).

Antibiotics 2023, 12, 941. https://doi.org/10.3390/antibiotics12050941 https://www.mdpi.com/journal/antibiotics

Antibiotics 2023, 12, 941 2 of 15

AMR of human pathogens is inter-linked with AMR of bacteria associated with

livestock, as well as the wider environment. Infections and resistance that originate in
humans, animals, foods and farm environments will inevitably lead to the dissemination
of infection with resistant bacteria and resistance genes in the wider environment [6,7].
This spreading of resistance may be facilitated by excreta coming into contact with soils
as well as surface and ground water [8]. The human acquisition of AMR from domestic
animals could occur by consuming contaminated animal-sourced foods, contaminated
water, contact with domestic animals or contact with contaminated environments [9–12].
Cross-species transfer of resistant bacteria or resistance genetic elements from animals
or the environment to humans has been reported [13–15]. The public health risks of a
possible transfer of resistant zoonotic agents from animals to humans led to policy changes,
such as a ban on the use of antibiotics as growth promoters in the European Union, and the
introduction of AMR monitoring systems in livestock food systems in many countries [16].
Sweden was the first country to ban the use of antibiotics for growth promotion in food
animal production in early 1986 [17]. Early insight about the risks of AMR was the main
reason for the decision [18].
E. coli is one of the most widespread bacteria throughout the world. It is a normal
commensal microbiota of the intestinal tract of animals and humans. However, not all
E. coli strains are harmless, as some are able to cause diseases in humans as well as in
mammals and birds [19,20]. For example, E. coli is a leading cause of extra-intestinal
infections with strains colonizing the gastrointestinal tract of patients [21]. Animals are
recognized as a reservoir for both human intestinal and extraintestinal pathogenic E. coli [22].
Enterohemorrhagic E. coli (EHEC), in particular EHEC O157:H7, a subtype of Shiga toxin
producing E. coli (STEC), is of particular concern. AMR has been reported in E. coli from
various animal species, the environment and in hospitalized patients from across the world,
with many strains exhibiting multi-drug resistance (MDR) [21]. The rapid emergence of
multi-drug-resistant pathogens has become one of the greatest concerns, as there are fewer,
or even sometimes no, effective antimicrobial agents available for infections caused by
these bacteria [23].
Given its widespread occurrence and capacity to assimilate resistances, E. coli has proved
useful as a sentinel for monitoring antimicrobial drug resistance in fecal bacteria [24]. Studying
AMR in E. coli is also important since the transmission of AMR through the environment
is probably more important in E. coli than any other member of the microbiota. E. coli can
remain viable in the environment (secondary habitat) for extended periods of time [25]. In
addition, most AMR in E. coli is encoded on mobile genetic elements that are transferable
between bacteria, thus enabling the rapid dissemination and maintenance of resistance genes
between bacteria of different species (horizontal transfer of AMR genes) [10,26–29]. Therefore,
AMR in E. coli is regarded as a major threat to public health [25].
The dynamics of AMR in developing countries are poorly understood, especially in
rural community settings, due to a lack of data on the prevalence of AMR and molecular
characteristics [21]. Although the surveillance capacity for AMR is minimal in most East
African countries, and current data on AMR patterns of common pathogenic bacteria
are sparse, high levels of AMR to commonly used antibiotics have been reported in this
region [1,30]. AMR in Ethiopia is also increasing at an increasing rate [31–34]. While
several separate studies have been conducted over the years on human patients, livestock,
foods and the environment, with results indicating that there is a danger of losing worthy
therapeutics due to the development of AMR by microorganisms, there are no robust
national antimicrobial susceptibility data to show trends. A recent systematic review and
meta-analysis revealed estimates of high AMR prevalence in bacteria from live animals,
foods of animal origin, food handlers and the environment [35].
Attempts have been made to identify E. coli strains, particularly O157:H7, in meat
and abattoir environments in Ethiopia and to understand its ecology and epidemiology,
including sources of contamination, prevalence and antibiotic resistance profiles [36–39].
Yet, the extent to which livestock feces can serve as a source of resistant E. coli is poorly
Antibiotics 2023, 12, 941 3 of 15

understood. However, this understanding is essential to develop effective strategies to

reduce the emergence and spread of resistance, which is a global priority. This paper
characterized the distribution of AMR of E. coli isolated from livestock feces and soil in a
low-resource, extensive smallholder livestock production system.

2. Results
2.1. Occurrence of E. coli and E. coli O157:H7
One or more E. coli was isolated in 131 (86.2%) cattle fecal samples, 140 (77.8%) sheep
samples, 89 (68.5%) goat samples and 51 (39.2%) soil samples.
The Biolog system (Omni Log ID) laboratory reader identified 42 (9.1%) of the 462 isolates
as E. coli O157:H7. Table 1 presents the occurrence of E. coli O157:H7 by sample types
and species of animals examined. The higher occurrence of E. coli O157:H7 was found
among isolates from goats (20.2%), cattle (11.4%) and soil samples (6.8%). Conversely, only
two isolates (1.4%) were characterized as E. coli O157:H7 from sheep samples.

Table 1. Occurrence of E. coli O157:H7 by sample types and species of animals examined.

Sample Type Positives (%) Odds Ratio p-Value CI for the Odds Ratio
Cattle feces 11.4 (7–18.1) 1.75 0.240 0.68–4.48
Sheep feces 1.4 (0.3–5.5) 0.19 0.045 0.04–0.96
Goat feces 20.2 (13.1–29.8) 3.4 0.009 1.36–8.68
Soil 6.8 (3.3–13.7) Ref

2.2. Occurrence of Antimicrobial Resistance in E. coli

Of 462 E. coli isolates tested, resistance to at least one antimicrobial was found in 51.9%
(43.4–60.3) of isolates from cattle, 33.6% (95% CI, 26.2–41.8) from sheep, 58.4% (95% CI,
47.9–68.2) from goats and in 52.9% (95% CI, 43.2–62.4) of soil samples, and a prevalence of
47.8 (95% CI, 43.3–52.4) across all livestock and soil samples combined was recorded.
The proportion of resistance to specific antibiotic classes among E. coli isolates from
cattle, sheep, goat and soil was generally low (Table 2). Among 440 isolates, 23.2% showed
resistance to amoxycillin/clavulanate, with 25.8% in cattle, 11.2% in sheep, 37.0% in goat
and 24.8% in soil samples. Among 451 isolates tested for tetracycline, overall, 8% were
resistant, with 3.9% resistance in cattle samples, 5.8% in sheep, 13.6% in goat and 11.3% in
soil isolates. A higher level of resistance against streptomycin was found in the 449 isolates
tested, with 33.2% of all samples testing positive, with 37.3% positive in cattle samples,
16.8% in sheep samples, 49.4% in goat samples and 36.4% in soil samples. A much smaller
proportion of isolates were resistant against the other antibiotics tested (Table 2).

Table 2. Percentages of E. coli isolates resistant to different antibiotic classes classified by sample type
(livestock spp. or soil).

Resistance Phenotypes in Different

Drug Class Antibiotics Host n Host Types
%* 95% Conf. Interval
Cattle 124 25.8 a 18.9–34.2
Sheep 134 11.2 b 6.9–17.7
Amoxycillin/clavulanate
Penicillin Goat 81 37.0 a 27.2–48
(AML10)
Soil 101 24.8 17.3–34.1
Total 440 23.2 19.4–27.4
Cattle 128 3.9 a 1.6–9.1
Sheep 138 5.8 2.9–11.2
Tetracyclines Tetracycline (TE30) Goat 88 13.6 b 7.9–22.5
Soil 97 11.3 6.4–19.4
Total 451 8 5.8–10.9
Antibiotics 2023, 12, 941 4 of 15

Table 2. Cont.

Resistance Phenotypes in Different

Drug Class Antibiotics Host n Host Types
%* 95% Conf. Interval
Cattle 115 0.9 0.1–5.9
Sheep 132 3 1.1–7.8
Doxycycline (DO30) Goat 67 0 -
Soil 95 3.2 1.1–9.3
Total 409 2 1–3.8
Cattle 127 2.4 0.7–7.1
Sheep 137 4.4 1.9–9.4
Fluoroquinolones Ciprofloxacin (CIP5) Goat 87 5.7 2.4–13.1
Soil 99 5.1 2.1–11.6
Total 450 4.2 2.7–6.5
Cattle 130 0a -
Sheep 136 2.2 0.7–6.6
Trimethoprim/Folate
Trimethoprim (W5) Goat 89 7.8 b 3.8–15.6
pathway inhibitors
Soil 101 4.9 2.1–11.4
Total 456 3.3 2–5.4
Cattle 127 0a -
Sheep 137 3.6 1.5–8.5
Sulfamethoxazole
Goat 87 9.1 b 4.6–17.4
trimethoprim (SXT25)
Soil 98 3.1 0.9–9.1
Total 449 3.6 2.2–5.7
Sulfonamide
Cattle 116 0.9 a 0.1–5.8
Sheep 133 1.5 ac 0.4–5.8
Sulfonamide (S3_300) Goat 69 7.2 3–16.3
Soil 93 8.6 b 4.4–16.3
Total 411 3.9 2.4–6.3
Cattle 122 4.9 2.2–10.5
Sheep 134 4.5 a 2–9.6
Gentamicin (CN10) Goat 82 14.6 b 8.5–24.1
Soil 101 10 5.4–17.5
Total 439 7.7 5.6–10.7
Aminoglycoside
Cattle 126 37.3 a 29.3–46.1
Sheep 137 16.8 b 11.4–24
Streptomycin (S25) Goat 87 49.4 ac 39.1–59.8
Soil 99 36.4 ad 27.5–46.3
Total 449 33.2 28.9–37.7
Cattle 117 0.9 0.1–5.8
Sheep 132 0 -
Cefuroxime (CXM30) Goat 74 4.1 13.1–11.9
Soil 94 2.1 0.5–8.1
Total 417 1.4 0.6–3.2
Cattle 115 8.7 4.7–15.4
Sheep 134 4.5 2.1–9.6
Cephalosporins Cefotaxime (CTX30) Goat 69 4.3 1.4–12.7
Soil 94 6.4 2.9–13.5
Total 412 6.1 4.1–8.8
Cattle 118 0.8 0.1–5.8
Sheep 133 0.8 1.1–5.2
Cefoxitin (FOX30) Goat 70 0
Soil 92 1.1 0.2–7.3
Total 413 0.7 0.2–2.2
Antibiotics 2023, 12, 941 5 of 15

Table 2. Cont.

Resistance Phenotypes in Different

Drug Class Antibiotics Host n Host Types
%* 95% Conf. Interval
Cattle 116 0 -
Sheep 137 1.5 0.3–5.7
Quinolones Nalidixic acid (NA30) Goat 72 1.4 0.2–9.2
Soil 93 3.2 1–9.6
Total 418 1.4 0.6–3.1
Cattle 124 1.6 0.4–6.2
Sheep 137 2.2 0.7–6.6
Chloramphenicol Chloramphenicol (C30) Goat 81 2.5 0.6–9.4
Soil 93 2.1 0.5–8.2
Total 435 2.1 1.1–3.9
Cattle 123 0 -
Sheep 132 0.8 0.1–5.2
Nitrofuran Nitrofurantoin (F300) Goat 83 2.4 0.6–9.2
Soil 101 1.9 0.4–7.6
Total 425 1.1 0.5–2.7
Each superscript letter (*) denotes a resistance phenotype percentage in different host types: the same letter or no
letter means the percentages do not differ significantly from each other at the 0.05 level (ANOVA).

2.3. Multiple Antimicrobial Resistance

Out of 375 isolates tested for resistance against all 15 antibiotics, 205 (54.7%) of the
isolates were susceptible to all of the antibiotics tested and 170 (45.3%) were resistant to at
least one antibiotic. However, 100 (26.7%) showed multiple drug resistance (resistant to
two or more antibiotics classes), with one sample resistant to 8 antibiotics classes. In total,
44 samples (11.8%) were resistant to ≥3 antibiotic classes, 18 (4.8%) were resistant to ≥4
and 11 (2.9%) were resistant to ≥5 (see Table 3). The most common co-resistant phenotype
observed was resistance to amoxycillin/clavulanate and streptomycin (18.8%).

Table 3. Multiple antimicrobial resistance of E. coli isolated from livestock and soil.

Number of
Predominant Resistance Profile
Antimicrobial No. of Isolates (%)
Composition *
Resistances
Zero - 205 (54.7)
One S; Aml; Ctx; Te; Cn 170 (45.3)
Two AmlS; SCtx; CipS; CnS; 56 (14.9)
Three AmlCnS; TeSS3; AmlTeS; AmlSF; AmlCipS 26 (6.9)
Four AmlCnSCxm; AmlCnTES 7 (1.9)
Five WamlTeSxtS; AmlCnSCDo 7 (1.9)
Six AmlCnSFCS3; AmlTeSxtCNaDo 3 (0.8)
Eight WamlCnTeCipSxtSS3 1 (0.3)
* Amoxycillin/clavulanate (Aml); Cefotaxime (Ctx); Cefoxitin (F); Cefuroxime (Cxm); Chloramphenicol (C);
Ciprofloxacin (Cip); Doxycycline (Do); Gentamicin (Cn); Nalidixic acid (Na); Streptomycin (S); Sulfamethoxazole
trimethoprim (Sxt); Sulfonamide (S3); Tetracycline (Te); Trimethoprim (W).

2.4. Antimicrobial Resistance among E. coli O157:H7 Isolates

Resistance among E. coli O157:H7 isolates was generally low regardless of the source
of isolation. Among the 42 isolates, 5 (17.2%) were resistant to streptomycin and 3 (10.3%)
were resistant to amoxycillin/clavulanate. Resistance to other antibiotics tested was rare,
with resistance to chloramphenicol (3.5%) and cefotaxime (3.5%) detected at low levels. All
isolates were susceptible to cefoxitin (second-generation cephalosporin).
Of all E. coli O157:H7 isolates, 19 (45.2%) showed MDR. The most common co-resistant
phenotype observed was to amoxycillin/clavulanate and streptomycin (20.7%).
Antibiotics 2023, 12, 941 6 of 15

2.5. Risk Factors for the Occurrence of Antimicrobial Resistance Phenotypes

Univariable analyses showed that six variables were potentially associated (p-value < 0.25)
with the occurrence of more than two or more antimicrobial resistance phenotypes in E. coli
isolated from livestock, and seven variables for E. coli isolated from soil.
Table 4 shows risk factors associated with the occurrence of ≥2 antimicrobial resistance
phenotypes among E. coli isolated from livestock using multivariable logistic regression
analysis. The variables ‘manure management’ and ‘what do you do with dead animals?’
were both highly correlated with agroecology and not used in the model. Agroecology
(pastoral system) and lack of access to professional animal health services were associated
with E. coli resistance for ≥2 antibiotics in the models. The odds of detecting E. coli resistance
to ≥2 antimicrobials in livestock fecal samples were nearly three times higher in lowland
pastoral production systems than in highland mixed crop–livestock production systems
(OR: 2.9; 95% CI, 1.72–5.17; p = 0.000).

Table 4. Potential predictor variables for the occurrence of ≥2 resistance phenotypes in livestock
using multivariable logistic regression analysis.

% AMR (95% Univariable Analysis Multivariable Analysis

Predictor Level/Category
CI) OR 95% CI p-Value OR 95% CI p-Value
Highland mixed
crop–livestock
Agroecology 7 (3.8–12.2) Ref
production system
(n = 158)
Pastoralist system
43.8 (35.5–52.5) 3.2 2.3–4.6 0.000 2.98 1.72–5.17 0.000
(n = 130)
Species mix Keep <3 species (n = 39) 41 (26.8–56.9) Ref
Keep ≥3 species (n = 244) 20.9 (16.2–26.5) 0.38 0.18–0.77 0.006 1.35 0.56–3.22 0.498
Leave on farm; Open air;
Manure mgt. Discard into environment 44.8 (36.3–53.6) Ref
(n = 125)
Used as fertilizer (n = 32) 15.6 (6.6–32.5) 0.23 0.09–0.68 0.004
Used for fuel (incl.
biogas); Sold for cash 4.8 (2.1–10.2) 0.06 0.04–0.20 0.000
(fuel) (n = 126)
Isolation of sick Yes (n = 150) 11.3 (7.1–17.5) Ref
animals
No (n = 133) 37.5 (29.7–46.1) 4.7 2.5–8.7 0.000 1.24 0.39–3.88 0.713
Allow mix of animals Yes (n = 95) 36.8 (27.7–46.9) Ref
on treatment
No (n = 188) 17 (12.3–23.1) 0.35 0.2–0.62 0.000 0.62 0.32–1.17 0.143
What do you do with
Leave as it is (n = 4) 75 (23.6–96.6) 22.2 1.73–59.1 0.010
dead animals?
Give to the dog (n = 112) 5.3 (2.4–11.4) 0.41 0.15–1.61 0.245
Bury (n = 42) 11.9 (5–25.6) Ref
Human consumption
42.4 (34–51.2) 4.84 1.81–12.9 0.002
(n = 125)
Access to professional
animal health Yes (n = 245) 22.8 (18.1–28.5) Ref
services/treatments
No (n = 38) 28.9 (16.8–45.2) 1.4 0.64–2.94 0.3
Access to regular
animal health services Yes (n = 256) 23.8 (18.9–29.4) Ref
(vaccination and
deworming)
No (n = 27) 22.2 (10.3–41.5) 0.91 0.35–2.36 0.537

Predictor variables for the occurrence of resistance to ≥2 antibiotics among E. coli

isolate in soil samples are shown in Table 5. Manure management was retained in this
model. Households that either used manure for fuel (incl. biogas) or sold manure for
cash had a reduction of 85% (OR: 0.15; 95% CI, 0.03–0.75; p = 0.03) and had higher odds
of having ≥2 antimicrobial resistance phenotypes in soil compared with those who left
manure either on the farm or in the open air or discarded manure into the environment.
Antibiotics 2023, 12, 941 7 of 15

Table 5. Potential predictor variables for the occurrence of ≥2 resistance phenotypes in soil using
multivariable logistic regression analysis.

% AMR (95% Bivariate Analysis Multivariable Analysis

Predictor Level/Category
CI) OR 95% CI p-Value OR 95% CI p-Value
Highland mixed
crop–livestock
Agroecology 24.1 (14.7–36.9) Ref
production system
(n = 58)
Pastoralist system
62.1 (43.3–77.8) 2.3 1.4–3.6 0.001
(n = 29)
Species mix Keep <3 species (n = 9) 55.5 (24.7–82.5) Ref
Keep ≥3 species (n = 77) 35.1 (25.1–46.4) 0.43 0.11–1.74 0.239
Leave on farm; Open air;
Manure mgt. Discard into environment 66.7 (47–81.8) Ref
(n = 27)
Used as fertilizer (n = 9) 44.4 (17.4–75.2) 0.4 0.08–1.86 0.243 0.47 0.12–1.885 0.291
Used for fuel (incl.
biogas); Sold for cash 20.4 (11.2–34.2) 0.13 0.04–0.36 0.000 0.15 0.03–0.75 0.03
(fuel) (n = 49)
Isolation of sick Yes (n = 52) 25 (14.9–38.6) Ref
animals
No (n = 34) 55.8 (38.9–71.6) 3.8 1.5–9.56 0.004 0.98 0.25–3.91 0.986
Allow mix of animals Yes (n = 23) 47.8 (28.5–67.7) Ref
on treatment
No (n = 63) 33.3 (22.7–45.9) 0.54 0.21–1.44 0.221
What do you do with (no
Leave as it is (n = 1) -
dead animals? observation)
Give to the dog (n = 47) 25.5 (14.9–40) 1.54 0.29–8.17 0.61
Bury (n = 11) 18.2 (4.5–51.3) Ref
Human consumption
66.7 (47–81.8) 9 1.59–50.7 0.013
(n = 27)
Access to professional
Yes (n = 75) 32 (22.3–43.4) Ref
animal health services
No (n = 11) 72.7 (40.9–91.1) 5.6 1.37–23.2 0.016 1.6 0.28–8.85 0.59
Access to regular
animal health services Yes (n = 76) 35.5 (25.4–47) Ref
(vaccination and
deworming)
No (n = 10) 50 (22.1–77.8) 1.81 0.48–6.83 0.378

3. Discussion
We found a similar prevalence of resistant E. coli in livestock fecal samples and soil.
Goat fecal samples had the highest prevalence of resistance (58%) to at least one antimi-
crobial in E. coli, followed by soil samples (53%). However, it is difficult to pinpoint the
origin of the antimicrobial resistance observed. The lowest proportion of resistance was
observed in isolates from sheep fecal samples. Despite the absence of drug stewardship in
the study area, the resistance level for individual antibiotics tested was generally low, with
a higher level of resistance against ‘older’ drugs such as streptomycin, followed by amoxy-
cillin/clavulanate and tetracycline. This is expected, as resistance in E. coli mainly occurs
against drugs that have been commonly used for farm animal treatments and/or prophy-
laxis for a long time [16,40–42]. Resistance against ‘newer’ drug classes (Cephalosporins,
Quinolones, Chloramphenicol, Nitrofuran) was lower. This is in agreement with a recent
systematic review and meta-analysis which noted that drug resistance in various samples,
including animal-sourced foods, was against older drugs such as ampicillin, amoxicillin,
streptomycin and tetracycline [35].
AMR occurrence in E. coli isolated from food-producing animals has been reported in
different countries, but due to differences in sampling strategies, isolation methods and
methods of AMR phenotype determination comparisons between studies is difficult. A
study in Kenya showed that E. coli resistance to aminoglycosides, sulfonamides, tetracy-
clines, trimethoprim and penicillin was high in both humans and livestock, while resistance
to cephalosporins and fluoroquinolones was low [43]. There is also evidence from com-
munity settings within countries in sub-Saharan Africa and in South Asia where E. coli
Antibiotics 2023, 12, 941 8 of 15

resistances to ‘older’ antimicrobials was common, with 65% of isolates resistant to ampi-
cillin, 67% to trimethoprim, 66% to trimethoprim/sulphamethoxazole, 56% to tetracycline
and 43% resistant to streptomycin [21]. In a study in the USA, out of 746 E. coli isolates
recovered from animal sources, 71.1% were resistant to tetracycline, 59% to streptomycin,
57.7% to sulfonamide and 34.1% to ampicillin [24].
Multi-drug-resistant pathogens have emerged worldwide [16]. In our study, the
prevalence of MDR in E. coli was 26.7% and the most common co-resistant phenotype
observed was to amoxycillin/clavulanate and streptomycin (18.8%). A relatively larger
proportion of MDR E. coli isolates was recovered from animals in a US study [24]. They
found that concurrent resistance to tetracycline and streptomycin was the most common co-
resistance phenotype (30%), followed by resistance to tetracycline and sulfonamide (29%).
In our study, 42 (9%) E. coli isolates from livestock feces and soil were E. coli O157:H7,
with higher proportions among isolates from goats and cattle. Hunduma (2018) also
reported comparable prevalence of 4.7% E. coli O157 in both milk and feces samples in cows
from a similar setting [44]. It is commonly cited that cattle are the primary reservoir of E. coli
O157:H7 [45], with small ruminants also implicated [46–48]. The resistance level among
E. coli O157:H7 isolates was generally low regardless of the source of isolation. Resistance to
streptomycin (17%) and amoxycillin/clavulanate (10.3%) were the most common resistance
profiles seen in these isolates. However, Hunduma found a higher level of E. coli O157
resistance to streptomycin (65%), tetracycline (59%) and Trimethoprim (24%) [44]. These
drugs are still commonly administered in humans [49] and animals [41,42] in Ethiopia.
Bekele et al. reported E. coli O157:H7 isolates from raw meat in Addis Ababa that were
resistant to different antibiotics including streptomycin (33%) and tetracycline (5%) [36].
With this study, we report for the first time AMR in E. coli O157:H7 isolated from soil, and
thus confirm that soil contaminated via feces can act as a source of drug-resistant microbial
pathogens including E. coli O157:H7. Greater attention should be paid to prevent E. coli
O157:H7 contamination of the human food chains, given its health impact. Many studies
have shown that the survival of E. coli O157:H7 in soil can lead to contamination of drinking
water, fruits and fresh vegetables and constitutes a major public health threat [9,50–54].
Furthermore, E. coli O157:H7 can cause severe hemorrhagic colitis and hemolytic uremia
in humans [55].
Our results also found higher odds for resistance in livestock from the lowland pastoral
production system. This may be due to higher infection pressure and probability of
recirculation of resistant isolates in the lowland agroecology and pastoral production
system. It is possible that warm temperatures offer more potential for bacteria to multiply,
with greater transference of antimicrobial resistance genes. Warmer temperatures are also
associated with higher insect populations, which can play a role in disseminating resistant
bacteria [56]. It could, however, also reflect the fact that improper use of antibiotics, mostly
without a proper diagnosis, is more common in these production systems [42]. Such
information is important to target AMR management practices.
Poor management, including the management and disposal of manure (i.e., leaving
manure either on the farm, or in the open-air or discarding manure into the environment)
was also strongly associated with detecting a higher level of E. coli resistance to more than
two or more antimicrobial resistance phenotypes in soil samples. Similarly, Muloi et al.
(2019) found that keeping manure inside the household compound was also significantly
associated with AMR carriage in humans [43]. Animal manure has been implicated as
a reservoir of AMR bacteria and AMR determinants [57,58]. Transmission of antibiotic-
resistant E. coli and resistance genes may also occur through environments contaminated
with feces, especially in developing countries [57,59].
Low levels of resistance are often overlooked, but can play an important role in the
expansion of resistance [60]. In these extensive smallholder and pastoral settings, there is
little to no testing of drug susceptibility during treatment of cases for both humans and
animals. Hence, minimizing resistance is crucial. There is a need to maintain an overview of
Antibiotics 2023, 12, 941 9 of 15

drug susceptibility through an AMR surveillance system that monitors resistance patterns
and trends.

4. Materials and Methods

4.1. Study Area
The study was conducted in two agroecological zones and production systems: (i) a
highland mixed crop–livestock production system (Menz Mama and Menz Gera district)
and (ii) a pastoral system (Yabello and Eleweya districts). Details of the characteristics
of the study area were published elsewhere [42]. Briefly, highland agroecology with a
mixed crop–livestock system is typical for areas above 2200 m above sea level (masl) in
which livestock husbandry depends on rain fed cropping. In lowland agroecology, pastoral
livestock production is widespread, with the community mainly dependent on livestock
and livestock products.
In recent publications, differences were reported between locations and production
systems in terms of characteristics of antimicrobial usage including: (1) access to antimicro-
bials, (2) types of antimicrobials used and (3) when they were used. Livestock producers
in mid/lowland pastoral systems appeared to use antibiotics more frequently than their
counterparts in highland and lowland mixed crop–livestock systems [42].

4.2. Study Design and Sample Size Determination

A cross-sectional study was conducted with 77 households selected from extensive
smallholder livestock systems in four districts. A total of 539 samples, which included
462 livestock fecal samples (cattle = 152, sheep = 180 and goats = 130) and 77 soil samples,
were collected.
For fecal samples, the number of animals to be sampled in the study was estimated by
the formula [61];
n = z2 × [Pexp (1 − Pexp)/d2] (1)
where z = 1.96, Pexp (the expected prevalence) = 0.11 and d = 0.05 (the desired level of
precision). Based on the result of a systematic review and meta-analysis, the overall pooled
prevalence of E. coli expected was 15% in all samples [62]. The required sample size was,
therefore, n = 231. To account for herd level clustering, the target sample size was adjusted
using an intra-cluster correlation coefficient of 0.2 with an average of 6 animals sampled
per herd. Accordingly, the design effect (D) of the study was calculated as 1.4 according to:

D = 1 + (m − 1) × ρ (2)

where m was cluster size (i.e., 6), ρ was 0.2 and the calculated sample size was adjusted by
multiplying by D. Therefore, the new sample size was 462 animals from 77 households.
Hence, 77 soil samples were collected from the homestead and barn areas of 77 households.
Household data were previously collected. In each household, details of household
demographics, farm characteristics, manure management, feed types, animal health con-
straints, disease prevention, animal health services, antimicrobial use and animal product
consumption were collected. Information on the selection of agroecological zones, districts
and villages and random household selection was described in [42].
Because this study does not focus on the number of isolates per animal, we restricted
the number of isolates to one or zero per animal.

4.3. Sample Collection and Pre-Enrichment Procedure

Fecal samples were taken from the rectum using a gloved hand and a sterile 50 milliliter
(mL) capacity Falcon tube.
Soil samples were collected from either the homestead or the barn area of ruminants,
from 2–5 cm beneath the surface. Approximately 10 g of soil, free of obvious fecal contami-
nation, was collected into sterile vials. If the surface of the area was not flat, samples were
collected from the lowest as well as middle and highest points, and mixed.
Antibiotics 2023, 12, 941 10 of 15

The fecal and soil samples were refrigerated and transported for laboratory analysis at
either Yabello Pastoral and Dryland Agricultural Research Center (for samples collected
from lowland pastoral areas) or the International Livestock Research Institute, Addis Ababa
(for samples from highland agroecology) within 4–6 h of collection.
Immediately upon arrival at the lab, a sample suspension was prepared using 1 g of
the sample in 9 mL of phosphate-buffered solution (5%). Samples were pre-enriched in
buffered peptone water and incubated at 370 ◦ C for 24 h.

4.4. Isolation and Identification of E. coli

A loop full of pre-enriched cultures was taken and inoculated on MacConkey agar
and then incubated at 37 ◦ C for 24 h. Typical colonies on MacConkey agar (pink, due to
their ability to ferment lactose) were Gram-stained and observed for their staining and
morphological characteristics, then transferred to eosin-methylene-blue (EMB) agar and
incubated for 24 h at 37 ◦ C. The colonies with metallic sheen on EMB agar, which is a
typical characteristic of E. coli, were then considered as E. coli-positive and transferred to
nutrient agar to be used for additional confirmatory biochemical tests (IMViC tests) and for
further identification for Biolog tests.
Presumptive pure E. coli isolates were further analyzed using the Biolog system
(OmniLog ID system, Hayward, CA, USA) following the standard procedures of the
manufacturer. Briefly, the purified cultures of E. coli were inoculated on BUG (Biolog
Universal Growth) agar medium; inoculums were prepared at a specified cell density
using inoculating fluid A (IF A); the Biolog microplate GEN III was inoculated with the
inoculums; the plate was incubated at 330 ◦ C for 22 h into the Ominilog apparatus; the
reaction pattern was entered; and results were obtained from the apparatus. The system
also further identified E. coli O157:H7 serotype.
The purified cultures of E. coli were then stored in glycerol added to TSB broth at
−200 ◦ C for further biotyping and other studies.

4.5. Antimicrobial Susceptibility Test

The antimicrobial susceptibility test on the isolates was performed according to the
National Committee for Clinical Laboratory Standards [63] using the Kibry-Bauer disk
diffusion test method on Muller-Hinton agar (Oxoid CM0337 Basingstoke, England).
From each isolate, four to five confirmed colonies grown on nutrient agar were trans-
ferred to a test tube of 5 mL Tryptone Soya Broth (TSB) (Oxoid). The broth culture was
incubated at 37 ◦ C for 18 h until growth reached the 0.5 McFarland turbidity standard.
Mueller-Hinton agar plates were readied according to the manufacturer’s guidelines
and held at room temperature for 30 min to allow drying. A sterile cotton swab was
dipped into the suspension and then swabbed uniformly in three directions over the
surface of Muller-Hinton agar plate (Oxoid Ltd, Hampshire, UK). After the plates dried,
antibiotic disks were placed using an automatic Oxoid antimicrobial disk dispenser onto the
inoculated plates and incubated at 37 ◦ C for 24 h. After incubation for 24 h, the diameter
of the zones of inhibition was measured and compared with zone size interpretation
guidelines described by the Clinical Laboratory Standard Institute [64] for the family
Enterobacteriaceae and determined as sensitive, intermediate or resistant.
The isolated E. coli were tested for sensitivity to the most commonly used antimi-
crobials. The zone of inhibition was interpreted based on the Performance Standards
for Antimicrobial Susceptibility Testing; Sixteenth Informational Supplement, as detailed
in Table 6.
Antibiotics 2023, 12, 941 11 of 15

Table 6. Zone interpretive chart for antimicrobials (inhibition zone diameter in mm).

Antimicrobial Agent Disc Content (µg) Resistant (≤) Intermediate Susceptible (≥)
Trimethoprim (W5) 5 10 11–15 16
Amoxycillin/clavulanate
10 13 14–17 18
(AML10)
Gentamicin (CN10) 10 12 13–14 15
Tetracycline (TE30) 30 11 12–14 15
Ciprofloxacin (CIP5) 5 15 16–20 21
Sulfamethoxazole
25 10 11–15 16
trimethoprim (SXT25)
Streptomycin (S25) 25 11 13–14 15
Cefuroxime (CXM30) 30 14 15–17 18
Nalidixic acid (NA30) 30 13 14–18 19
Chloramphenicol (C30) 30 12 13–17 18
Cefotaxime (CTX30) 30 22 23–25 26
Cefoxitin (FOX30) 30 14 15–17 18
Doxycycline (DO30) 30 10 11–13 14
Sulfonamide (S3_300) 300 12 13–16 17
Nitrofurantoin (F300) 300 14 15–16 17

4.6. Data Analysis

Overall and host- and sample-specific (livestock and soil) distribution of resistance
phenotypes were determined by dividing the number of isolates with specific resistance
phenotypes by the total number of isolates examined. Difference in proportions of samples
in a group with resistance to one or more antibiotics was determined using Chi-squared
tests and one-way analysis of variance (ANOVA; soil vs. different livestock groups).
Bonferroni’s multiple-comparison test was performed post hoc for pairwise comparisons
between groups, and p-values < 0.05 were considered significant.
Host- and sample-specific occurrence of E. coli O157:H7 were determined by dividing
the number of E. coli O157:H7 isolated in each host by the total number of samples examined.
The proportions of antimicrobial resistance were also determined by dividing the number
of E. coli O157:H7 isolates with specific resistance phenotypes by the total number of
isolates examined.
From the household data, eight variables were used as potential predictor vari-
ables for the occurrence of AMR. The variables were agroecology (highland mixed crop–
livestock production system vs. pastoralist system); species mix (keep <3 livestock species
vs. keep ≥3 livestock species); manure management (leave on farm or open air, discard
into environment vs. use as fertilizer vs. use for fuel (incl biogas), sale for cash); isolation
of sick animals (yes vs. no); allow mix of animals on treatment (yes vs. no); what do you
do with dead animals (leave as it is vs. give to the dog vs. bury vs. human consumption);
access to professional animal health services/treatments (yes vs. no); access to regular
animal health services/vaccination and deworming (yes vs. no). A subset of the data that
only included isolates tested for all antimicrobials (n = 288 for livestock and n = 87 for
soil) was used for analysis of risk factors. To identify risk factors for having resistance
to ≥2 antibiotics, odds ratios (ORs) were calculated using univariable logistic regression,
followed by multivariable logistic regression.
Separate models were developed for livestock and soil, and a forward selection method
with a significance level of 5% was used to select a suitable model. Models were adjusted
for the cluster effect using robust standard error estimation for cluster sampling. When
selecting important variables to the model, the Wald statistic associated with the variable
was used instead of the likelihood ratio test (LRT) statistic [65].
Collinearity was assessed pair-wise via calculation of Spearman rank correlations
between predictors. Variables with a p-value < 0.25 were considered for multivariable
analysis, provided that there was no collinearity (r < |0.7|) between them. Potential
confounders were considered in every model as variables that, if present, changed the
Antibiotics 2023, 12, 941 12 of 15

coefficient for one or more significant variables by an amount that was important to report,
taken as 10% [66]. All variables with a p-value ≤ 0.05 were retained in the final model.
There were no biologically plausible interactions between the main effects expected and
tested. Data were analyzed using Stata software version 16 (College Station, TX, USA).

5. Conclusions and Recommendations

These findings provide insights into the status of resistance in livestock and soil, and
associated risk factors in low resource settings in Ethiopia. AMR patterns for E. coli from
livestock and soil showed similarities. The resistance level for individual antibiotics tested
was generally low, with the highest prevalence of resistance detected against streptomycin
(33%), followed by amoxycillin/clavulanate (23%) and tetracycline (8%). The most common
co-resistant phenotype observed was against amoxycillin/clavulanate and streptomycin.
The findings showed that soil contaminated via feces can act as a source of drug-resistant
microbial pathogens including E. coli O157:H7. The results also revealed that agroecology
and production systems and manure management were strongly associated with occurrence
of resistance. Additional molecular analysis of resistance genes is now planned to further
investigate evidence of overlapping patterns of transferable resistance genes between
livestock and soil. This would provide more definitive data on how resistance gene clusters
have evolved and the context in which genes are maintained in the absence of known
selection pressures.
In conclusion, we recommend that animal health and biosecurity practices, such as
manure management, are radically improved, and that an integrated AMR surveillance
system is established.

Author Contributions: Conceptualization, B.A.G., B.W., H.D.W. and K.A.; Methodology, B.A.G.,
B.W., T.J.D.K.-J. and K.A.; Formal Analysis, B.A.G.; Investigation, B.A.G., M.T., A.K., S.A., A.O.,
H.D.W. and G.A.; Data Curation, B.A.G., M.T., A.K., A.O. and S.A.; Visualization, B.A.G., T.J.D.K.-J.
and K.A.; Writing—Original Draft Preparation, B.A.G.; Writing—Review and Editing, B.A.G., B.W.,
T.J.D.K.-J., G.A. and K.A.; Supervision, B.W. and K.A.; Project Administration, B.A.G., B.W. and
H.D.W.; Funding Acquisition, B.W. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was conducted under CRP livestock and continued under the CGIAR
Initiative Sustainable Animal Productivity for Livelihoods, Nutrition and Gender (SAPLING). CGIAR
research is supported by contributions from the CGIAR Trust Fund. CGIAR is a global research
partnership for a food-secure future dedicated to transforming food, land and water systems in a
climate crisis. The German Academic Exchange Service (DAAD) supported the project through a
ILRI-DAAD Doctoral Fellowship for the first author. The funders played no role in the design or
conclusion of the study and any opinions, findings, conclusions and recommendations expressed
here are those of the authors alone.
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Review Board (or Ethics Committee) of Addis Ababa University, College of Veterinary Medicine and
Agriculture (Certificate Ref. No.: VM/ERC/01/07/10/2018).
Informed Consent Statement: Not applicable. Written informed consent for participation was not
required for this study in accordance with the national legislation and the institutional requirements.
Data Availability Statement: The datasets generated for this study are available on request to the
corresponding author.
Acknowledgments: The authors thank the Animal Health Institute (AHI) and Yabello Regional
Veterinary Laboratory in Ethiopia and their technical staff, who were involved in field data col-
lection and laboratory analysis. The farmers and pastoralists who participated in the study are
greatly appreciated.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
Antibiotics 2023, 12, 941 13 of 15

References
1. World Health Organization. GLASS|Global Antimicrobial Resistance Surveillance System (GLASS) 2020; World Health Organization
(WHO): Geneva, Switzerland, 2020.
2. Iwu-Jaja, C.J.; Jaca, A.; Jaja, I.F.; Jordan, P.; Bhengu, P.; Iwu, C.D.; Okeibunor, J.; Karamagi, H.; Tumusiime, P.; Fuller, W.; et al.
Preventing and managing antimicrobial resistance in the African region: A scoping review protocol. PLoS ONE 2021, 16, e0254737.
[CrossRef] [PubMed]
3. World Bank. Drug-Resistant Infections: A Threat to Our Economic Future; World Bank: Washington, DC, USA, 2017.
4. Jasovsky, D.; Littmann, J.; Zorzet, A.; Cars, O. Antimicrobial Resistance; S. Karger AG: Basel, Swtizerland, 2010; Volume 6.
5. Tang, K.L.; Caffrey, N.P.; Nóbrega, D.B.; Cork, S.C.; Ronksley, P.E.; Barkema, H.W.; Polachek, A.J.; Ganshorn, H.; Sharma, N.;
Kellner, J.D.; et al. Restricting the use of antibiotics in food-producing animals and its associations with antibiotic resistance in
food-producing animals and human beings: A systematic review and meta-analysis. Lancet Planet. Health 2017, 1, e316–e327.
[CrossRef] [PubMed]
6. Lepper, H.C.; Woolhouse, M.E.J.; van Bunnik, B.A.D. The Role of the Environment in Dynamics of Antibiotic Resistance in
Humans and Animals: A Modelling Study. Antibiotics 2022, 11, 1361. [CrossRef]
7. Woolhouse, M.; Ward, M.; van Bunnik, B.; Farrar, J. Antimicrobial resistance in humans, livestock and the wider environment.
Philos. Trans. R. Soc. B Biol. Sci. 2015, 370, 20140083. [CrossRef] [PubMed]
8. Berglund, B. Environmental dissemination of antibiotic resistance genes and correlation to anthropogenic contamination with
antibiotics. Infect. Ecol. Epidemiol. 2015, 5, 28564. [CrossRef]
9. Mukherjee, A.; Cho, S.; Scheftel, J.; Jawahir, S.; Smith, K.; Diez-Gonzalez, F. Soil survival of Escherichia coli O157:H7 acquired by a
child from garden soil recently fertilized with cattle manure. J. Appl. Microbiol. 2006, 101, 429–436. [CrossRef]
10. Berg, E.S.; Wester, A.L.; Ahrenfeldt, J.; Mo, S.S.; Slettemeås, J.S.; Steinbakk, M.; Samuelsen, Ø.; Grude, N.; Simonsen, G.S.;
Løhr, I.H.; et al. Norwegian patients and retail chicken meat share cephalosporin-resistant Escherichia coli and IncK/bla CMY-2
resistance plasmids. Clin. Microbiol. Infect. 2017, 23, 407.e9–407.e15. [CrossRef]
11. Johnson, J.R.; Clabots, C.; Kuskowski, M.A. Multiple-Host Sharing, Long-Term Persistence, and Virulence of Escherichia coli
Clones from Human and Animal Household Members. J. Clin. Microbiol. 2008, 46, 4078–4082. [CrossRef]
12. Ercumen, A.; Pickering, A.J.; Kwong, L.H.; Arnold, B.F.; Parvez, S.M.; Alam, M.; Sen, D.; Islam, S.; Kullmann, C.; Chase, C.; et al.
Animal Feces Contribute to Domestic Fecal Contamination: Evidence from E. coli Measured in Water, Hands, Food, Flies, and Soil
in Bangladesh. Environ. Sci. Technol. 2017, 51, 8725–8734. [CrossRef]
13. Sivagami, K.; Vignesh, V.J.; Srinivasan, R.; Divyapriya, G.; Nambi, I.M. Antibiotic usage, residues and resistance genes from food
animals to human and environment: An Indian scenario. J. Environ. Chem. Eng. 2020, 8, 102221. [CrossRef]
14. Young, C.C.W.; Karmacharya, D.; Bista, M.; Sharma, A.N.; Goldstein, T.; Mazet, J.A.K.; Johnson, C.K. Antibiotic resistance genes
of public health importance in livestock and humans in an informal urban community in Nepal. Sci. Rep. 2022, 12, 13808.
[CrossRef] [PubMed]
15. Moser, K.A.; Zhang, L.; Spicknall, I.; Braykov, N.P.; Levy, K.; Marrs, C.F.; Foxman, B.; Trueba, G.; Cevallos, W.; Goldstick, J.; et al.
The Role of Mobile Genetic Elements in the Spread of Antimicrobial-Resistant Escherichia coli From Chickens to Humans in
Small-Scale Production Poultry Operations in Rural Ecuador. Am. J. Epidemiol. 2018, 187, 558–567. [CrossRef] [PubMed]
16. Ma, F.; Xu, S.; Tang, Z.; Li, Z.; Zhang, L. Use of antimicrobials in food animals and impact of transmission of antimicrobial
resistance on humans. Biosaf. Health 2021, 3, 32–38. [CrossRef]
17. Maron, D.F.; Smith, T.J.; Nachman, K.E. Restrictions on antimicrobial use in food animal production: An international regulatory
and economic survey. Glob. Health 2013, 9, 48. [CrossRef]
18. Björkman, I.; Röing, M.; Sternberg Lewerin, S.; Stålsby Lundborg, C.; Eriksen, J. Animal Production With Restrictive Use of
Antibiotics to Contain Antimicrobial Resistance in Sweden—A Qualitative Study. Front. Vet. Sci. 2021, 7, 619030. [CrossRef]
19. Dho-Moulin, M.; Fairbrother, J.M. Avian pathogenic Escherichia coli (APEC). Vet Res. 1999, 30, 299–316.
20. Kaper, J.B.; Nataro, J.P.; Mobley, H.L.T. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2004, 2, 123–140. [CrossRef]
21. Ingle, D.J.; Levine, M.M.; Kotloff, K.L.; Holt, K.E.; Robins-Browne, R.M. Dynamics of antimicrobial resistance in intestinal Es-
cherichia coli from children in community settings in South Asia and sub-Saharan Africa. Nat. Microbiol. 2018, 3, 1063–1073. [CrossRef]
22. Bélanger, L.; Garenaux, A.; Harel, J.; Boulianne, M.; Nadeau, E.; Dozois, C.M. Escherichia coli from animal reservoirs as a potential
source of human extraintestinal pathogenic E. coli. FEMS Immunol. Med. Microbiol. 2011, 62, 1–10. [CrossRef]
23. 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]
24. Tadesse, D.A.; Zhao, S.; Tong, E.; Ayers, S.; Singh, A.; Bartholomew, M.J.; McDermott, P.F. Antimicrobial Drug Resistance in
Escherichia coli from Humans and Food Animals, United States, 1950–2002. Emerg. Infect. Dis. 2012, 18, 741–749. [CrossRef] [PubMed]
25. Barrera, S.; Cardenas, P.; Graham, J.P.; Trueba, G. Changes in dominant Escherichia coli and antimicrobial resistance after 24 h in
fecal matter. MicrobiologyOpen 2019, 8, e00643. [CrossRef]
26. Johnson, T.J.; Logue, C.M.; Johnson, J.R.; Kuskowski, M.A.; Sherwood, J.S.; Barnes, H.J.; DebRoy, C.; Wannemuehler, Y.M.;
Obata-Yasuoka, M.; Spanjaard, L.; et al. Associations Between Multidrug Resistance, Plasmid Content, and Virulence Potential
Among Extraintestinal Pathogenic and Commensal Escherichia coli from Humans and Poultry. Foodborne Pathog. Dis. 2012, 9,
37–46. [CrossRef] [PubMed]
Antibiotics 2023, 12, 941 14 of 15

27. Liu, Y.-Y.; Wang, Y.; Walsh, T.R.; Yi, L.-X.; Zhang, R.; Spencer, J.; Doi, Y.; Tian, G.; Dong, B.; Huang, X.; et al. Emergence of
plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: A microbiological and molecular
biological study. Lancet Infect. Dis. 2016, 16, 161–168. [CrossRef] [PubMed]
28. Dobiasova, H.; Dolejska, M. Prevalence and diversity of IncX plasmids carrying fluoroquinolone and β-lactam resistance genes in
Escherichia coli originating from diverse sources and geographical areas. J. Antimicrob. Chemother. 2016, 71, 2118–2124. [CrossRef]
29. Wright, G.D. The antibiotic resistome: The nexus of chemical and genetic diversity. Nat. Rev. Microbiol. 2007, 5, 175–186. [CrossRef]
30. Ampaire, L.; Muhindo, A.; Orikiriza, P.; Mwanga-Amumpaire, J.; Bebell, L.; Boum, Y. A review of antimicrobial resistance in East
Africa. Afr. J. Lab. Med. 2016, 5, a432. [CrossRef]
31. Berhe, D.F.; Beyene, G.T.; Seyoum, B.; Gebre, M.; Haile, K.; Tsegaye, M.; Boltena, M.T.; Tesema, E.; Kibret, T.C.; Biru, M.; et al.
Prevalence of antimicrobial resistance and its clinical implications in Ethiopia: A systematic review. Antimicrob. Resist. Infect.
Control 2021, 10, 168. [CrossRef]
32. Ibrahim, R.A.; Teshal, A.M.; Dinku, S.F.; Abera, N.A.; Negeri, A.A.; Desta, F.G.; Seyum, E.T.; Gemeda, A.W.; Keficho, W.M.
Antimicrobial resistance surveillance in Ethiopia: Implementation experiences and lessons learned. Afr. J. Lab. Med. 2018,
7, a770. [CrossRef]
33. Moges, F.; Endris, M.; Mulu, A.; Tessema, B.; Belyhun, Y.; Shiferaw, Y.; Huruy, K.; Unakal, C.; Kassu, A. The growing challenges of
antibacterial drug resistance in Ethiopia. J. Glob. Antimicrob. Resist. 2014, 2, 148–154. [CrossRef]
34. Seboxa, T.; Amogne, W.; Abebe, W.; Tsegaye, T.; Azazh, A.; Hailu, W.; Fufa, K.; Grude, N.; Henriksen, T.-H. High Mortality
from Blood Stream Infection in Addis Ababa, Ethiopia, Is Due to Antimicrobial Resistance. PLoS ONE 2015, 10, e0144944.
[CrossRef] [PubMed]
35. Gemeda, B.A.; Assefa, A.; Jaleta, M.B.; Amenu, K.; Wieland, B. Antimicrobial resistance in Ethiopia: A systematic review and meta-
analysis of prevalence in foods, food handlers, animals, and the environment. One Health 2021, 13, 100286. [CrossRef] [PubMed]
36. Bekele, T.; Zewde, G.; Tefera, G.; Feleke, A.; Zerom, K. Escherichia coli O157:H7 in Raw Meat in Addis Ababa, Ethiopia: Prevalence
at an Abattoir and Retailers and Antimicrobial Susceptibility. Int. J. Food Contam. 2014, 1, 4. [CrossRef]
37. Abreham, S.; Teklu, A.; Cox, E.; Sisay Tessema, T. Escherichia coli O157:H7: Distribution, molecular characterization, antimicrobial
resistance patterns and source of contamination of sheep and goat carcasses at an export abattoir, Mojdo, Ethiopia. BMC Microbiol.
2019, 19, 215. [CrossRef]
38. Abebe, M.; Hailelule, A.; Abrha, B.; Nigus, A.; Birhanu, M.; Adane, H.; Genene, T.; Daniel, H.; Getachew, G.; Merga, G.; et al.
Antibiogram of Escherichia coli strains isolated from food of bovine origin in selected Woredas of Tigray. Ethiop. J. Bacteriol. Res
2014, 6, 17–22. [CrossRef]
39. Messele, Y.E.; Abdi, R.D.; Yalew, S.T.; Tegegne, D.T.; Emeru, B.A.; Werid, G.M. Molecular determination of antimicrobial resistance
in Escherichia coli isolated from raw meat in Addis Ababa and Bishoftu, Ethiopia. Ann. Clin. Microbiol. Antimicrob. 2017,
16, 55. [CrossRef]
40. White, D.G. Antimicrobial Resistance in Pathogenic Escherichia coli from Animals. In Antimicrobial Resistance in Bacteria of Animal
Origin; ASM Press: Washington, DC, USA, 2019; pp. 145–166.
41. Beyene, T.; Endalamaw, D.; Tolossa, Y.; Feyisa, A. Evaluation of rational use of veterinary drugs especially antimicrobials and
anthelmintics in Bishoftu, Central Ethiopia. BMC Res. Notes 2015, 8, 482. [CrossRef]
42. Gemeda, B.A.; Amenu, K.; Magnusson, U.; Dohoo, I.; Hallenberg, G.S.; Alemayehu, G.; Desta, H.; Wieland, B. Antimicrobial Use
in Extensive Smallholder Livestock Farming Systems in Ethiopia: Knowledge, Attitudes, and Practices of Livestock Keepers.
Front. Vet. Sci 2020, 7, 55. [CrossRef]
43. Muloi, D.; Kiiru, J.; Ward, M.J.; Hassell, J.M.; Bettridge, J.M.; Robinson, T.P.; van Bunnik, B.A.D.; Chase-Topping, M.; Robertson, G.;
Pedersen, A.B.; et al. Epidemiology of antimicrobial-resistant Escherichia coli carriage in sympatric humans and livestock in a
rapidly urbanizing city. Int. J. Antimicrob. Agents 2019, 54, 531–537. [CrossRef]
44. Hunduma, D. Isolation, Identification and Antimicrobial Susceptibility Test of Escherichia coli O157 and Non-Typhoid Salmonella
Species in Milk and Faeces of Lactating Cows and Camels in Borana Pastoral Area, Southern Ethiopia. Master’s Thesis, College
of Veterinary Medicine and Agriculture of Addis Ababa University, Bishoftu, Ethiopia, 2018.
45. Munns, K.D.; Selinger, L.B.; Stanford, K.; Guan, L.; Callaway, T.R.; McAllister, T.A. Perspectives on Super-Shedding of Escherichia
coli O157:H7 by Cattle. Foodborne Pathog. Dis. 2015, 12, 89–103. [CrossRef]
46. Pao, S.; Patel, D.; Kalantari, A.; Tritschler, J.P.; Wildeus, S.; Sayre, B.L. Detection of Salmonella Strains and Escherichia coli
O157:H7 in Feces of Small Ruminants and Their Isolation with Various Media. Appl. Environ. Microbiol. 2005, 71, 2158–2161.
[CrossRef] [PubMed]
47. Gencay, Y.E. Sheep as an important source of E. coli O157/O157:H7 in Turkey. Vet. Microbiol. 2014, 172, 590–595. [CrossRef] [PubMed]
48. Mersha, G.; Asrat, D.; Zewde, B.M.; Kyule, M. Occurrence of Escherichia coli O157:H7 in faeces, skin and carcasses from sheep and
goats in Ethiopia. Lett. Appl. Microbiol. 2010, 50, 71–76. [CrossRef] [PubMed]
49. Bitew, A.; Adane, A.; Abdeta, A. Bacteriological spectrum, extended-spectrum β-lactamase production and antimicrobial
resistance pattern among patients with bloodstream infection in Addis Ababa. Sci. Rep. 2023, 13, 2071. [CrossRef]
50. Zhang, T.; Wang, H.; Wu, L.; Lou, J.; Wu, J.; Brookes, P.C.; Xu, J. Survival of Escherichia coli O157:H7 in Soils from Jiangsu Province,
China. PLoS ONE 2013, 8, e81178. [CrossRef]
51. Ogden, I.; Fenlon, D.R.; Vinten, A.J.; Lewis, D. The fate of Escherichia coli O157 in soil and its potential to contaminate drinking
water. Int. J. Food Microbiol. 2001, 66, 111–117. [CrossRef]
Antibiotics 2023, 12, 941 15 of 15

52. Franz, E.; Semenov, A.V.; Termorshuizen, A.J.; de Vos, O.J.; Bokhorst, J.G.; van Bruggen, A.H.C. Manure-amended soil characteris-
tics affecting the survival of E. coli O157:H7 in 36 Dutch soils. Environ. Microbiol. 2008, 10, 313–327. [CrossRef]
53. Yao, Z.; Wei, G.; Wang, H.; Wu, L.; Wu, J.; Xu, J. Survival of Escherichia coli O157:H7 in Soils from Vegetable Fields with Different
Cultivation Patterns. Appl. Environ. Microbiol. 2013, 79, 1755–1756. [CrossRef]
54. Duffitt, A.D.; Reber, R.T.; Whipple, A.; Chauret, C. Gene Expression during Survival of Escherichia coli O157:H7 in Soil and Water.
Int. J. Microbiol. 2011, 2011, 340506. [CrossRef]
55. Boyce, T.G.; Swerdlow, D.L.; Griffin, P.M. Escherichia coli O157:H7 and the Hemolytic–Uremic Syndrome. N. Engl. J. Med. 1995,
333, 364–368. [CrossRef]
56. Collignon, P.; Beggs, J.J.; Walsh, T.R.; Gandra, S.; Laxminarayan, R. Anthropological and socioeconomic factors contributing to global
antimicrobial resistance: A univariate and multivariable analysis. Lancet Planet. Health 2018, 2, e398–e405. [CrossRef] [PubMed]
57. Graham, D.W.; Knapp, C.W.; Christensen, B.T.; McCluskey, S.; Dolfing, J. Appearance of β-lactam Resistance Genes in Agricultural
Soils and Clinical Isolates over the 20th Century. Sci. Rep. 2016, 6, 21550. [CrossRef] [PubMed]
58. Udikovic-Kolic, N.; Wichmann, F.; Broderick, N.A.; Handelsman, J. Bloom of resident antibiotic-resistant bacteria in soil following
manure fertilization. Proc. Natl. Acad. Sci. USA 2014, 111, 15202–15207. [CrossRef] [PubMed]
59. Pehrsson, E.C.; Tsukayama, P.; Patel, S.; Mejía-Bautista, M.; Sosa-Soto, G.; Navarrete, K.M.; Calderon, M.; Cabrera, L.; Hoyos-
Arango, W.; Bertoli, M.T.; et al. Interconnected microbiomes and resistomes in low-income human habitats. Nature 2016, 533,
212–216. [CrossRef] [PubMed]
60. American Society for Microbiology. Antibiotic Resistance: An Ecological Perspective on an Old Problem; American Society for
Microbiology: Washington, DC, USA, 2009. Available online: https://www.ncbi.nlm.nih.gov/books/NBK559361/ (accessed on
18 November 2022).
61. Thrusfield, M. Veterinary Epidemiology; Blackwell Scientific Publications: Oxford, UK, 1995.
62. Assefa, A.; Bihon, A. A systematic review and meta-analysis of prevalence of Escherichia coli in foods of animal origin in Ethiopia.
Heliyon 2018, 4, e00716. [CrossRef] [PubMed]
63. NCCLS—National Committee for Clinical Laboratory Standards. Methodology for the Serum Bactericidal Test, 2nd ed.; Tentative
Guideline—NCCLS Document M21-T; NCCLS: Villanova, PA, USA, 1990.
64. CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 32nd ed.; CLSI Supplement M100; CLSI: St. Louis, MO,
USA, 2022.
65. Jayatillake, R.V.; Sooriyarachchi, M.R.; Senarathna, D.L.P. Adjusting for a cluster effect in the logistic regression model: An
illustration of theory and its application. J. Natl. Sci. Found. Sri Lanka 2011, 39, 201. [CrossRef]
66. Lee, P.H.; Burstyn, I. Identification of confounder in epidemiologic data contaminated by measurement error in covariates. BMC
Med. Res. Methodol. 2016, 16, 54. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.