Journal of Global Antimicrobial Resistance 36 (2024) 284–292

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Journal of Global Antimicrobial Resistance

journal homepage: www.elsevier.com/locate/jgar

Risk factors associated with mcr-1 colistin-resistance gene in

Escherichia coli broiler samples in northern Jordan
Mohammad H. Gharaibeh a,∗, Sahba Y. Al Sheyab a, Shawkat Q. Lafi b, Eman M. Etoom a
a
Department of Basic Veterinary Medical Science, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid, Jordan
b
Department of Pathology and Public Health, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid, Jordan

a r t i c l e i n f o a b s t r a c t

Article history: Objectives: The purpose of this study was to determine the prevalence of colistin-resistant Escherichia coli
Received 30 June 2023 carrying mcr-1, and to identify risk factors associated with mcr gene-mediated resistance.
Revised 6 January 2024
Methods: In total, 385 cloacal samples were collected from 125 broiler farms and a questionnaire con-
Accepted 10 January 2024
taining information about each farm was designed and filled.
Available online 6 February 2024
Results: Most of the antibiotics used in the disk diffusion method were highly resistant in all sam-
Editor: Stefania Stefani ples, with tetracycline and penicillin showing 100% and 99.7% resistance, respectively. Additionally, avian
Keywords: pathogenic E. coli (APEC) virulence genes frequency and percentage of APEC were identified, including
E. coli sitA, iucC, and astA at 77%, 70.5%, and 62% respectively. In total, 214 of 360 isolates were positive for
Antimicrobial resistance APEC (59.4%). Based on the minimum inhibitory (MIC) test, 58% of the isolates (n = 209 of 360) were
Colistin resistance resistant to colistin, with 39.7% displaying the mcr-1 gene. The statistical analysis of risk factors that in-
Virulence genes fluence colistin resistance prevalence revealed several significant factors, including commercial feed, farm
Broiler management, sanitization, and antibiotic use. Irregular health checks for workers, non-dipping of feet be-
Risk factors
fore entering poultry houses, and the use of commercial poultry feeds all contributed to higher levels of
colistin resistance as measured by MIC. On the other hand, doxycycline and commercial feed was 4 and
3.2 times more likely to occur based on the final logistic model of the mcr-1 gene, respectively.
Conclusion: Our results suggest that better biosecurity protocols should be implemented in poultry farms
to reduce antibiotic-resistant bacteria. Additionally, antibiotics should be carefully monitored and used
only when necessary.
© 2024 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial
Chemotherapy.
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

1. Introduction in the host and the environment. Virulence factors are usually en-
coded on pathogenicity islands (PAIs), plasmids and other mobile
Escherichia coli are divided into extraintestinal pathogenic E. genetic elements, and these factors help E. coli cause systemic dis-
coli (ExPEC) and commensal E. coli [1]. The ExPEC strains include ease in the host [3]. A major contributor to APEC is stress, which
uropathogenic E. coli (UPEC), newborn meningitis-causing E. coli results in its translocation to extra-intestinal sites [4]. The com-
(NMEC), and avian pathogenic E. coli (APEC), which are endemic bination of tension, low ventilation, reduction of feed or water,
and severely impact the poultry industry, causing chronic respira- high temperature, and overcrowding favours the growth of col-
tory disorders, septicaemia, salpingitis, omphalitis, avian colibacil- ibacillosis [5]. Colibacillosis can be significantly reduced by re-
losis, and embryonic deaths. To date, more than 150 genes have ducing the amount of E. coli bacteria in the flock’s water and
been identified as being involved in APEC virulence factors [2], feed, sanitizing the environment, and providing good air qual-
having different roles in adhesion, invasion, colonization, biofilm ity and litter quality [6]. Birds feeding can affect the severity
formation, motility, intracellular survival, cell lysis and damage, of colibacillosis [7]. For instance, a high-protein diet, high sele-
metal acquisition for growth, bacterial persistence, and resistance nium ratio, and more vitamins A and E result in enhanced im-
mune systems. Numerous strategies are currently being employed
to reduce the mortality and incidence associated with APEC infec-
∗
Corresponding author. tion in poultry, including antimicrobial therapy with tetracyclines,
E-mail address: [email protected] (M.H. Gharaibeh). cephalosporins, sulfonamides, or quinolones [8] and vaccinations

https://doi.org/10.1016/j.jgar.2024.01.003
2213-7165/© 2024 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
M.H. Gharaibeh, S.Y.A. Sheyab, S.Q. Lafi et al. Journal of Global Antimicrobial Resistance 36 (2024) 284–292

against the various serotypes [9]. Colistin has been used to treat dia Eosin Methylene Blue [EMB] [Oxoid, England] and E. coli Hi-
infections caused by carbapenemase-producing Enterobacteriaceae. Chrome [HI Media Laboratories Pvt. Ltd, Mumbai, India]) within
Consequently, the global increase in such species has increased the few hours of collection and incubated for 18–24 h at 37°C. The sus-
use of colistin [10]. Colistin has been banned by both the Euro- pected colonies (greenish colonies on agar) were subcultured again
pean Medicines Agency and the Chinese Ministry of Veterinarians on the same selective Hi-chrome E. coli media, and then incubated
as an animal feed additive [11]. Resistance to colistin is primar- for 18–24 hours at 37°C. Gram stain, oxidase test, and standard bio-
ily caused by chromosomal mutations that modify the target site chemical test were used to confirm the results. Confirmed samples
where colistin can make electrostatic interactions with Lipid A of were preserved in cryostat tubes at –70°C in 20% glycerol with 80%
lipopolysaccharides of the outer membrane [12]. Various members Muller Hinton broth [25].
of the Enterobacteriaceae family, including Klebsiella Spp., E. coli,
Salmonella Spp., and Enterobacter Spp., have acquired colistin resis-
2.4. Antimicrobial susceptibility test using standard disc diffusion
tance through the mobilization of colistin resistance genes (mcr)
method
[13], which are plasmid-mediated genes that were discovered for
the first time in 2015 by Liu et al. [14]. Later, the genes were de-
All confirmed samples as E. coli were tested by the agar
tected in livestock sources in 57 countries and in the animals that
disk diffusion method using Muller-Hinton agar (Oxoid, Eng-
produce food in 57 countries [15]. The critical cause of polymyxin
land) according to the Clinical and Laboratory Standards Insti-
resistance is mcr gene-coding products that alter lipid A of the LPS
tute (CLSI) guidelines. Each E. coli sample was tested for 20 dif-
core [16]. The mcr-1 gene was first identified in E. coli isolated from
ferent antimicrobial disks (Oxoid, England); cephalexin (30 μg),
poultry in the 1980s [17]. Because E. coli is the most prevalent
penicillin (10 μg), ciprofloxacin (5 μg), doxycycline (30 μg), aztre-
mcr-positive bacteria, it could be considered both a carrier and a
onam (30 μg), imipenem (10 μg), gentamycin (10 μg), florfeni-
spreader of mcr. Among the mcr genes, mcr-1 is most prevalent,
col (30 μg), kanamycin (5 μg), tigecycline (15 μg), cefepime
followed by mcr 2–10. By paying more attention to mcr-positive
(30 μg), amoxicillin-clavulanate (20/10 μg), cefoxitin (30 μg),
isolates from humans, higher rates may be detected than in ani-
sulphamethoxazole-trimethoprim (23.7/1.25 μg), chloramphenicol
mals [15]. The presence of multiple carbapenemases and extended-
(30 μg), tetracycline (30 μg), fosfomycin (50 μg), meropenem (10
spectrum bacterial resistance genes on the same plasmid or on dif-
μg), amoxicillin (10 μg), and nalidixic-acid (30 μg) [26]. The disc
ferent plasmids is a common occurrence, which leads to the spread
diffusion test defines bacteria as susceptible, intermediate, or re-
of multidrug resistance [18–20]. ExPEC strains that are found in
sistant [27].
humans and birds are very similar. It is highly probable that birds
will zoonotically transmit the mcr-1 gene to humans [21]. This may
occur as a result of infected food or direct contact with an animal 2.5. Minimum inhibitory concentration for colistin susceptibility test
[22]. As another threat, pathogenic bacteria can exchange genetic
material and plasmid encoded resistance genes with intestinal mi- The minimum inhibitory concentrations (MICs) were deter-
crobiota, a process known as horizontal gene transfer that spreads mined by the broth microdilution method in a cation-adjusted
virulence and resistance [23]. In the coming years, the spread of Mueller–Hinton II broth (CA-MHBII) for the colistin antibiotic, in
resistant bacteria and resistance genes is expected to increase [24]. accordance with the CLSI guidelines. The raw colistin antibiotic
Therefore, this study observed multidrug-resistant E. coli in faeces was obtained in its pure form (DADvet, Jordan). Colistin resistance
from healthy broiler chickens in Jordan and investigated their viru- was determined using the 2017 European Committee on Antibac-
lence, resistance to colistin, and mcr-carrying isolates; additionally, terial Susceptibility Testing (EUCAST) criteria [28].
we identified risk factors associated with mcr-mediated resistance.

2.6. Identification of colistin resistance genes by multiplex PCR

2. Materials and methods
All isolated samples were screened by multiplex PCR targeting
2.1. Ethical approval
five mcr genes (mcr 1–5) [29]. The reaction mixture contained 3 μL
of DNA template, 0.5 μL of each forward and reverse primer, 5 μL
This study was reviewed and approved by the Animal Care and
of 5x HOT FIREPol® Blend Master Mix (Solis BioDyne, Tartu, Esto-
Use Committee (ACUC) of the Jordan University of Science and
nia), and 16 μL of nuclease-free water, with a final concentration of
Technology (JUST- ACUC).
25 μL (Supplementary Table S1). The DNA amplification was per-
formed on a (BIO-RAD) thermocycler with an initial denaturation
2.2. Sample collection
at 94°C for 15 min, 25 cycles for denaturation at 94°C for 30 s an-
nealing at 58°C for 90 s, and extension at 72°C for 1 min, followed
A total of 385 faecal samples were collected from healthy
by final extension at 72°C for 10 min.
broiler chickens from 125 farms from four governorates (Irbid,
Ajloun, Jerash, and Al-Mafraq) in northern Jordan (Fig. 1). In a per-
sonal interview with each farm owner, a questionnaire survey was 2.7. Molecular identification for 16 APEC virulence genes
filled out regarding farming data. The questionnaire covered topics
such as farm location and building design, water and feed sources, All E. coli isolates were screened by two multiplex PCRs to de-
biosecurity, management, diseases, vaccines, and antimicrobial us- tect 16 virulence genes [30]; the first one targeted 9 virulence
age (Appendix A). All faecal samples were brought to the Micro- genes and the second targeted 7 virulence genes (Supplementary
biology laboratory at JUST, Faculty of Veterinary Medicine, Depart- Table S2) [31]. The DNA amplification for both reactions was per-
ment of Basic Veterinary Medical Sciences within eight hours after formed on a (BIO-RAD) thermocycler (3 μL of DNA template, 0.5
collection using an icebox. μL of each forward and reverse primer, 5 μL of 5x HOT FIREPol®
Blend Master Mix, and 16 μL of nuclease-free water). The initial
2.3. E. coli isolation and identification denaturation at 94°C for 3 min, 25 cycles of denaturation at 94°C
for 30 s annealing at 63°C for the first reaction and 58°C for the
Cotton swabs from the cloaca of 385 birds were collected and second reaction for 30 s, extension at 68°C for 3 min, followed by
transferred onto transport media (Escherichia coli-selective me- a final extension at 72°C for 10 min.

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Fig. 1. Geographical location of the sample collection area. Three hundred and eighty-five chicken faecal samples were collected from different areas in northern Jordan:
Irbid (1), Ajloun (2), Jerash (3), and Al-mafraq (4) governorates.

2.8. Statistical analysis (60.3%), followed by Al-Mafraq, with 36.3% and 24.8%, respectively.
Jerash was the third area, with 6.4% and 3.3%, followed by Ajloun,
Based on the farm owner’s answers, data regarding sample cir- with 4.5% and 11.5%. Table 1 illustrates the distribution of farm lo-
cumstances were collected and analysed using SPSS version 24 cations and the number and percentage of farms that had positive
(SPSS Corp., IBM, Armonk, NY). Initially, associations between an- E. coli samples and APEC samples in relation to colistin resistance
timicrobial resistance among all 360 E. coli samples (MICs and mcr by MIC and those harbouring mcr-1 gene.
outcome variables) and the hypothesized risk factors (based on the
literature) were screened using univariate analysis (χ 2 test with a 3.2. Antimicrobial susceptibility results
confidence interval [CI] of 95%). Potential risk factors with P ≤ 0.05
(χ 2 test; two-sided test) and no collinearity (r ≤ 0.6) were con- Most antibiotics showed high resistance, including tetracy-
sidered in building the final multivariate logistic regression model cline (100%), penicillin (99.7%), amoxicillin (99.2%), chlorampheni-
using Wald backward logistic regression analysis. The final model col (98.8%), and florfenicol (98.1%). However, meropenem (1.9%)
was performed and tested to fit the Hosmer and Lemeshow-of-fit and imipenem (2.2%) displayed the lowest resistance levels. Addi-
test. tional details can be found in Figure 2 and Table 2.

3. Results 3.3. Colistin resistance

Among the 385 samples tested, 360 were positive for E. coli. The MIC of 360 E. coli samples was conducted using a colistin
Samples were collected from 125 poultry farms in various geo- antimicrobial agent. The results were interpreted in accordance
graphical regions of northern Jordan (Table 1, Fig. 1). Irbid has the with EUCAST as resistant ≥2 mg\L (Supplementary Table S3). In
highest percentage of poultry farms (52.8%) and colistin resistance total, 209 E. coli samples were colistin resistant (58%), while 151

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Table 1
Distribution of farm locations, number and percentage of farms, Escherichia coli samples, and APEC samples in relation to colistin resistance by minimum inhibitory concen-
tration (MIC) and those harbouring the mcr-1 gene.

Area # and % of broiler Samples # and +ve E. coli samples APEC samples # Colistin resistance
farms (n = 125) % (n = 385) and % (n = 360) and % (n = 214)
+MIC % –MIC + mcr % –mcr %
n = 209 n = 151 n = 83 n = 277

Irbid 66 (52.8%) 236 (61.3%) 211 (58.6%) 123 (57.5%) 126 (60.3%) 85 (56.3%) 57 (68.7%) 154 (55.6%)
Al-mafraq 46 (36.8%) 100 (26%) 100 (27.8%) 62 (29%) 52 (24.8%) 48 (31.8%) 8 (9.6%) 92 (33.2%)
Jerash 8 (6.4%) 21 (5.5%) 21 (5.8%) 16 (7.5%) 7 (3.3%) 14 (9.3%) 3 (3.6%) 18 (6.5%)
Ajloun 5 (4%) 28 (7.2%) 28 (7.8%) 13 (6.1%) 24 (11.5%) 4 (2.6%) 15 (18.1%) 13 (4.7%)

Table 2
Percentage and number of antimicrobial resistant Escherichia coli samples from broiler faecal samples using disc diffusion method.

Antimicrobial (abbreviation) Disc content (μg) Disk diffusion interpretive criteria (mm) E. coli (360)
Cloacal samples
R S Number and percent of resistant samples

β -lactams
Penicillin (p) 10 μg ≤14 >15 359 (99.7%)
Amoxicillin (AML) 10 μg ≤22 >26 357 (99.2%)
Azetronem (ATM) 30 μg ≤17 >21 126 (34.9%)
Imipenem (IPM) 10 μg ≤19 >23 8 (2.2%)
Meropenem (MEM) 10 μg ≤19 >23 7 (1.9%)
β -lactamase inhibitors
Amoxicillin-clavulanic acid (AMC) 20/10 μg ≤13 >18 139 (38.5%)
Tetracyclines
Tetracycline (TE) 30 μg ≤11 >15 360 (100%)
Doxycycline (Do) 30 μg ≤10 >10 347 (96.1)
Tigcycline (TGC) 15 μg ≤15 >18 89 (24.7%)
Sulfonamides
Sulphamethoxazole_trimethoprim (SXT) 23.7/1.25 μg ≤10 >16 346 (96.1%)
Fluoroquinolones
Nalidixic acid (NA) 30 μg ≤13 >19 351 (97.5%)
Ciprofloxacin (CIP) 5 μg ≤15 >21 347 (96.4%)
Aminoglycosides
Kanamycin (K) 5 μg ≤13 >18 339 (94.2%)
Gentamycin (CN) 10 μg ≤12 >15 205 (57.1%)
Cephalosporins
Cefepime (FEB) 30 μg ≤18 >25 63 (17.5%)
Cefoxitin (FOX) 30 μg ≤14 >18 56 (15.8%)
Cephalexin (CL) 30 μg ≤12 >18 256 (71.2%)
Phosphoric acid derivatives
Fosfomycin (FOS) 50 μg ≤12 >26 209 (58.1%)
Phenicols
Chloramphenicol (C) 30 μg ≤12 >18 356 (98.8%)
Florfenicol (FFC) 30 μg ≤10 >16 353 (98.1%)

Fig. 2. Percentage of antimicrobial resistance of Escherichia coli samples by disk diffusion method against 20 antimicrobial agents.

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Table 3
Percentage and number of antimicrobial resistant Escherichia coli samples from broiler faecal samples using disc diffusion method in association to colistin-resistant (mcr-1)
positive Escherichia coli samples, statistically significant at P ≤ 0.05 χ 2 (two-sided test).

Antimicrobial (abbreviation) Disc content (μg) E. coli (360) # and % of Colistin resistance mcr-1 (n = 83) χ2
Cloacal samples resistant samples Samples # and % P value of mcr -1
n = 360
R S R S

β -lactams
penicillin (p) 10 μg ≤14 >15 359 (99.7%) 83 (100%) 0 (0.0%) 0.583
Amoxicillin (AML) 10 μg ≤22 >26 357 (99.2%) 82 (98.7%) 1 (1.2%) 0.671
Azetronem (ATM) 30 μg ≤17 >21 126 (34.9%) 32 (38.5%) 51(61.4%) 0.438
Imipenem (IPM) 10 μg ≤19 >23 8 (2.2%) 3 (3.6%) 80 (96.4%) 0.326
Meropenem (MEM) 10 μg ≤19 >23 6 (1.9%) 2 (2.4%) 81 (97.6%) 0.546
β -lactamase inhibitors 38 (45.7%) 45(54.2%)
Amoxicillin-clavulanic acid (AMC) 20/10 μg ≤13 >18 140 (38.8%) 0.141
Tetracyclines
Tetracycline (TE) 30 μg ≤11 >15 360 (100%) 83 (100%) 0 (0.0%) -a
Doxycycline (Do) 30 μg ≤10 >10 346(96.1) 79(95.2%) 4(4.8%) 0.617
Tigecycline (TGC) 15 μg ≤15 >18 90 (25%) 26 (31.3%) 57 (68.7%) 0.129
Sulfonamides
Sulphamethoxazole_ 23.7/1.25 μg ≤10 >16 346 (96.1%) 79 (95.2%) 4 (4.8%) 0.617
trimethoprim (SXT)
Fluoroquinolones
nalidixic acid (NA) 30 μg ≤13 >19 352 (97.8%) 82(98.8%) 1 (1.2%) 0.473
Ciprofloxacin (CIP) 5 μg ≤15 >21 347 (96.1%) 82(98.8%) 1 (1.2%) 0.149
Aminoglycosides
Kanamycin (K) 5 μg ≤13 >18 339 (94.2%) 79 (95.2%) 4 (4.8%) 0.653
Gentamycin (CN) 10 μg ≤12 >15 204 (56.6%) 46 (55.4%) 37 (44.6%) 0.794
Cephalosporines
Cefepime (FEB) 30 μg ≤18 >25 62 (17.2%) 13 (15.7%) 70 (84.3%) 0.667
Cefoxitin (FOX) 30 μg ≤14 >18 55 (15.3%) 9(10.8%) 74(89.2%) 0.200
Cephalexin (CL) 30 μg ≤12 >18 256 (71.2%) 54(65.1%) 29 (34.9%) 0.165
Phosphoric acid derivatives
Fosfomycin (FOS) 50 μg ≤12 >26 211(58.6%) 43 (51.8%) 40(48.2%) 0.151
Phenicol
Chloramphenicol (C) 30 μg ≤12 >18 356 (98.8%) 83 (100%) 0 (0.0%) 0.270
Florfenicol (FFC) 30 μg ≤10 >16 353 (98.1%) 80(96.4%) 3(3.6%) 0.209

NOTE: P value: χ 2 test value for the difference between antimicrobial resistant samples and colistin resistant (MIC and mcr) E. coli samples, statically significant at P ≤ 0.05
(two-sided test).
MIC, minimum inhibitory concentration; R, resistant samples; S, sensitive samples.
a
No statistics are computed because production is a constant.

(42.4%) were susceptible to colistin. The mcr-1–5 genes have been northern Jordan. By using a χ 2 test at a CI of 95%, the colistin-
identified in 209 MIC-resistant E. coli samples against colistin (Sup- resistance results of the MIC and mcr-1 of the same samples were
plementary Fig. S1). According to the results, 83 (39.7%) samples compared. Four risk factors were identified for colistin resistance
were found to contain mcr-1 (the only type detected), while 126 by MIC, including open poultry houses, the source of one-day-old
(60.3%) were found to be lacking the gene (Table 1). Based on the chickens, feeding, the presence of other animals on the farm, steril-
statistical analysis of resistant samples against 20 antimicrobials by ization, and antibiotic treatment (Supplementary Table S6). In con-
disc diffusion test and colistin resistance by MIC and mcr-1 PCR trast, seven risk factors were found to be significantly related to
analysis, there were no significant relationships between colistin the distribution and faecal shedding of colistin-resistant E. coli har-
resistance and other antimicrobial resistance (Table 3, Supplemen- bouring the mcr-1 gene, including farm organization, sterilization,
tary Table S4). and antibiotic therapy (Supplementary Table S7). The final logistic
model was constructed using both the colistin resistance MIC and
3.4. Analysis of the APEC virulence genes the mcr-1. According to the final logistic regression model, three
risk factors were associated with colistin resistance tested by MIC:
To determine the prevalence of APEC, 16 APEC virulence genes using commercial feed, regular inspections on the farm, and foot
(VAGs) were tested by PCR on 360 E. coli samples (Supplemen- dipping prior to entering poultry houses (Table 4); whereas only
tary Figs. S2 and S3). The sitA gene had the highest number of two risk factors were associated with colistin resistance tested by
positive samples (n = 278), followed by iucC (n = 254), and astA mcr-1 gene: commercial feed and doxycycline antibiotic (Table 5).
(n = 224). In contrast, the lowest percentage (0.6%) was found for The incidence of colistin resistance in broiler farms is 3–5 times
the genes hlyD, kpslll, and papC (n = 2). On a total of 360 samples, higher when commercial feed is consumed in both MIC and mcr-1
214 (59.4%) were identified as APECs. Specifically, one sample had E. coli; it is ca. 6 times more likely to be detected on farms without
9 VAGs, compared to 13 with 8 VAGs, and 64 with 7 VAGs, 59 with a regular worker’s checkup, 2 times more on farms without a foot
6 VAGs, and 77 with 5 VAGs (Supplementary Table S5). dip, and 4 times more on farms that use doxycycline.

3.5. Risk factors and statistical analysis 4. Discussion

Based on questionnaire data from 125 poultry farms, statistical Escherichia coli is a common commensal bacteria found in
analysis was conducted to identify risk factors. Most of the samples the gastrointestinal tracts of poultry. In this study, E. coli had a
were collected from healthy chickens at different farm locations in prevalence rate of 93.5%, similar to that in nearby countries like

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Table 4
Results of multivariable logistic regression modeling combination of colistin resistance MIC in 125 poultry farms.

Method Variable B SE Wald df sig OR

MIC Commercial feed 1.590 0.586 7.358 1 .007 4.905

Workers regular 1.859 0.612 9.218 1 .002 6.419
checkup
Foot dip .881 0.482 3.346 1 .067 2.414

-2 log likelihood = 130.05

Hosmer and Lemeshow Test χ 2 test =1.368 at P < 0.85
B, log-odd; CI, confidence interval; OR, odd ratio at CI 95%; SE, standard error.

Table 5
Results of multivariable logistic regression modeling of colistin resistance mcr-1 in 125 poultry farms.

Method Variable B SE Wald df sig OR

mcr-1 Commercial feed 1.18 0.41 8.33 1 0.004 3.26

Doxycycline use 1.40 0.55 6.60 1 0.01 4.06

-2 log likelihood = 149.11

Hosmer and Lemeshow Test χ 2 test =0.206 at P < 0.85
B, log-odd; CI, confidence interval; OR, odd ratio at CI 95%; SE, standard error.

Lebanon, with 97% from fresh faecal samples [32], The prevalence bacteriostatic drug when used in combination with colistin [45,46].
rate was higher than those in Saudi Arabia (60%) [33], Qatar (52%) A multivariable logistic regression analysis revealed that the use of
[34], and Iraq (45%) [35]. The presence of E. coli was previously doxycycline as an antibiotic treatment was statistically associated
detected in 53.4% of sick chickens in Jordan using both conven- with a higher risk of developing mcr-1 (Table 3). Disk diffusion
tional methods and the RapIDTM ONE System [31]. 59.4% of the testing for antibiotic resistance did not show a significant associ-
E. coli samples in this study were APEC isolates that had five or ation with colistin resistance, nor with MIC or mcr-1 colistin re-
more virulence genes. In this study, 16 APEC VAGs were examined sistance (Table 3, Supplementary Table S4). However, it is obvious
that are most associated with E. coli pathogenicity that causes col- that there is a collinearity, as the percentages of resistant samples
ibacillosis in birds. A similar prevalence of APEC (68.1%) has been are very similar between colistin and other antimicrobials. A total
noted in developed countries [36]. It is anticipated that the iden- of 55 E. coli samples (98.2%) were resistant to at least three antimi-
tification of these virulence factors will contribute to the develop- crobial groups, so they were classified as multidrug-resistant [47].
ment of new treatment options [37]. In terms of virulence genes, Collinearity between resistance percentages and high prevalence of
sitA accounted for 77.2% of the total, followed by iucC 70.5%, both mcr-1 can be explained by resistance genes to different groups of
of which are involved in iron acquisition, and astA 62.2%, which is antimicrobials could exist together in the same mobile element
responsible for producing toxins [2,38]. The findings of this study [26], especially genes encoded for fluoroquinolones, carbapene-
are similar to those of a study conducted in Jordan in 2019, in mases and β -lactamase resistance, suggesting they are simulta-
which APEC were isolated and analysed from sick chickens. In this neously co-transferred with colistin resistant genes such as mcr-1
study, sitA, iucC, and astA were the predominant genes [31]. VAGs [20,48]. The tetracycline antibiotic was not recommended for the
have been using as genetic markers to study genetic similarity treatment of E. coli infections in poultry farms, and its resistance
and phylogenetic studies between different E. coli isolates. There may be attributed to its use as a growth promoter [49,50]. In con-
were several virulence genes identified in this study sample (iucD, trast, the low level of resistance to meropenem (2.2%), imipenem
cvi/cva, ibeA, tsh, iss, irp2, and vat) and these genes are commonly (1.9%), cefoxitin (17.2%), and cefepime (15.3%) in poultry is not sur-
found in isolates from APEC, UPEC, and NMEC [39]. In contrast, prising given that these antibiotics are difficult to obtain due to
(sfa, hlyA, and ibeA) were found to be specific to UPEC and NMEC their high cost [51,52]. Several strains containing the mcr genes 1
[40], which might account for their low prevalence in APEC iso- and 2 have been isolated from humans, animals that produce food,
lates. PapC, iss, and ibeA genes have been detected in human ExPEC and the environment [53]. There is a high prevalence of colistin re-
and UPEC (growth in human urine), as well as in ExPEC isolates sistance in Jordanian poultry (39.7%), which indicates the serious-
from chicken samples [41,42]. Avian pathogenic E. coli and human ness of the problem of antibiotic misuse, particularly in the case of
ExPEC may share virulence genes, along with antimicrobial resis- colistin. Until this study, there were no previous studies in Jordan
tance genes, that might be transferred via plasmids, transposons, investigating the presence of the mcr genes in the animal sector,
or integrons into humans [43]. In developing countries like Jordan, and therefore the current study is significant because it is the first
where there are few or no programs or systems to monitor antibi- study in Jordan to demonstrate that the mcr-1 gene is present in
otic use, the use of antibiotics in farm animals has received little E. coli bacteria isolated from poultry. It should be noted that only
attention [44]. During this study, E. coli isolates were tested against one Jordanian study detected the mcr-1 gene in Klebsiella pneumo-
20 antibiotics that represent the most used antibiotics in poultry niae bacteria isolated from humans [54]. In this regard, it is im-
farm production and management. Among the most used antibi- portant to consider the experiences of other countries in this area,
otics, tetracycline, penicillin, amoxicillin, chloramphenicol, nalidixic especially China, as the country is the largest producer and con-
acid, and ciprofloxacin showed the highest rates of antibiotic re- sumer of colistin, consuming more than 90% of its production [14].
sistance, with 100%, 99.7%, 99.2%, 98.8%, 97.8%, and 96.1%, respec- Due to this, a great deal of research has been conducted regarding
tively (Table 2). It is common practice in the poultry industry to MSR genes in China. The prevalence of the MSR gene in China is
use tetracyclines and penicillins (Hassan et al., 2021). In a previous much lower than in Jordan, especially in recent years when some
study [31] conducted with aseptic swabs from sick birds in Jordan, studies have shown a prevalence of only 2% [55,56]. This decrease
the resistance to amoxicillin was lower (93.3%). According to this in resistance rate seems to be directly related to the ban on col-
study, amoxicillin was administered to chickens at 38/125 farms, istin in the animal sector; before the implementation of the col-
contributing to their high resistance. Doxycycline is an effective istin ban (i.e. in 2016 and before), 17.8% E. coli isolates from the

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M.H. Gharaibeh, S.Y.A. Sheyab, S.Q. Lafi et al. Journal of Global Antimicrobial Resistance 36 (2024) 284–292

Chinese poultry sector were found to carry the mcr-1 gene. Dur- virulence genes (VAGs). Our findings indicate that broiler chickens
ing the ban period (May 2017–May 2023), 335 (12.4%) of 2689 are potential reservoirs for antibiotic-resistant APEC, which may
E. coli isolates were positive for the mcr-1 gene (Anyanwu et al., result in major public health implications for individuals in di-
2023). In our region, a recent study in the Gaza Strip, Palestine, rect or indirect contact with them. There were also multiple risk
suggests that the ban on colistin use in the animal sector at the factors strongly associated with higher colistin resistance in this
end of 2018 is also having a positive effect, as the mcr-1 gene has study. Therefore, it is essential to develop strategies that will re-
decreased significantly in the percentage of pathogenic E. coli iso- duce the risk of antibiotic resistance in poultry farming. This in-
lated from turkey flocks in the Gaza Strip [57]. As an opportunistic cludes the implementation of biosecurity measures, vaccination
pathogen, APEC can only translocate to extra-intestinal sites when programs, and the judicious use of antimicrobials to reduce the
stressed [58]. This can be attributed to increased host susceptibil- spread of antibiotic-resistant bacteria.
ity, which can occur when there is a co-infection and poor wel-
fare. In this study, most poultry farms that participated are open, Funding: This work was supported by the Deanship of Research,
have less than 10,0 0 0 birds, and have less than 10 houses on the Jordan University of Science and Technology, Irbid, Jordan (Re-
farm, which may exclude environmental stress caused by crowding search Grant No:4 /2020).
[59–61] (although the type of house [opened or closed] has been
significantly associated with colistin resistance by mcr-1). Further- Competing interests: None declared
more, most chickens (110/125) live with other animals on the same
farm, which might cause stress to the birds, as well as transmit Ethical approval: This study was reviewed and approved by the
other infections to them, which has been shown to be an impor- Animal Care and Use Committee (ACUC), Jordan University of Sci-
tant risk factor associated with colistin resistance in mcr-1 and ence and Technology (JUST- ACUC).
MIC. There is no doubt that poor sterilization can be a primary
reason for spreading infection and disease outbreaks [62]. When Supplementary materials
hygiene is poor and control measures are not practiced, antimicro-
bial resistant strains spread more rapidly [63]. According to Singer Supplementary material associated with this article can be
et al. [64], it has been observed that mcr-1 gene is present in one found, in the online version, at doi:10.1016/j.jgar.2024.01.003.
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