Antimicrobial resistance profile of Escherichia coli isolated from poultry litter

M. J. Khong ,* A. M. Snyder,* A. K. Magnaterra,* M. M. Young ,y N. L. Barbieri,y and

S. L. Weimer *,z,1
*
Department of Animal and Avian Sciences, University of Maryland, College Park, MD, 20742, USA; yDepartment of
Population Health, University of Georgia College of Veterinary Medicine, Athens, GA, 30602, USA; and zDepartment
of Poultry Science, University of Arkansas, Fayetteville, AR, 72701, USA

ABSTRACT Antimicrobial resistance is a threat to (27.9%), streptomycin (29.4%), ampicillin (20.6%),

animal and human health. As a commensal and zoo- colistin (13.2%), sulphonamides (8.8%), and imipenem
notic bacterium, Escherichia coli has the potential to (1.5%). Multidrug resistance to at least 3 antimicrobials
be a pathogenic source of antimicrobial resistance. The was observed in 22.1% of isolates. The identified
purpose of this study aimed to investigate the antimi- O-types of the E. coli isolates were O15, O75, O78, and
crobial resistance profile of E. coli isolated from litter O91. There was a greater likelihood that the genes
collected from pens in a broiler chicken experiment. groEL, aph(3)IA, silP, sull, aadA, qacEdelta1, iroN,
E. coli was isolated from litter samples (n = 68 ompTp, and hlyF were present in isolates that exhibited
isolates) of 16 pens housing broilers to d 53 of age. ampicillin resistance (P ≤ 0.05). There was a greater
Resistance to 10 antimicrobials was observed by disc likelihood that the groEL gene was present in isolates
diffusion. The presence of 23 antimicrobial and heavy resistant to ampicillin, colistin, tetracycline, sulphona-
metal resistance genes, O serogroups, and avian patho- mides, or cephalothin (P ≤ 0.05). Further characteriz-
genic E. coli (APEC-like) minimal predictor genes were ing E. coli antimicrobial resistance is essential and aids
identified through PCR. E. coli isolates presented the in developing effective solutions, thereby furthering the
greatest resistance to cephalothin (54.4%), tetracycline One Health objective.
Key words: Escherichia coli, APEC, poultry litter, antimicrobial resistance, disc diffusion
2023 Poultry Science 102:102305
https://doi.org/10.1016/j.psj.2022.102305

INTRODUCTION treatment and increase mortality in humans and ani-

mals (Munita and Arias, 2016).
Antimicrobial resistance is one of the most important Commonly found as a commensal (nonpathogenic)
global health issues because it affects human and animal organism, E. coli can become pathogenic by acquiring
populations (World Health Organization, 2019). The U. virulence factors on plasmids or other mobile genetic ele-
S. Food and Drug Administration reported that 2.8 mil- ments via horizontal gene transfer, thus enabling certain
lion people in the United States contracted an antimi- strains of E. coli to cause intestinal or extraintestinal
crobial-resistant infection in 2019, leading to over 35,000 disease (Kaper et al., 2004; de Oliveira et al., 2020). A
deaths (United States Food and Drug Administra- pathotype of concern in the poultry industry is avian
tion, 2019). The One Health concept arises from the pathogenic E. coli (APEC), which is the etiological
intersections of animals, humans, and the environment agent of colibacillosis and can manifest as infections
and aims to attain optimal public health by preventing such as airsacculitis, polyserositis, and septicemia (Dho-
and controlling zoonotic diseases (OHITF. One Health Moulin and Fairbrother, 1999). Colibacillosis negatively
Initiative Task Force 2008; Bidaisee and Macpher- impacts the health and welfare of poultry and incidence
son, 2014). Infections caused by antimicrobial-resistant can be related to the quality of feed, water and litter,
bacteria can exacerbate existing complications in antimicrobial use and stewardship, and management
practices (Nolan et al., 2013). Morbidity from E. coli
infections can increase the use of antimicrobials for ther-
Ó 2022 The Authors. Published by Elsevier Inc. on behalf of Poultry apeutic treatment, resulting in significant economic
Science Association Inc. This is an open access article under the CC BY losses for poultry producers. Mounting evidence suggests
license (http://creativecommons.org/licenses/by/4.0/). that APEC-contaminated poultry is a source of extrain-
Received July 15, 2022.
Accepted October 27, 2022. testinal pathogenic E. coli causing human disease
1
Corresponding author: sweimer@uark.edu (Rodriguez-Siek et al., 2005; Vincent et al., 2010;

1
2 KHONG ET AL.

Manges and Johnson, 2012; Manges, 2016). In a study September to November 2020. The birds were not vacci-
comparing the incidences of antimicrobial resistance nated or treated with any antimicrobials. All animal
across food products (such as vegetable salads and raw procedures were approved by the University of Mary-
meats), raw chicken was reported to have the highest E. land Institutional Care and Use Committee (protocol
coli incidence (23.3%) of antimicrobial resistance #R-JUL-20-35). Briefly, 400 day-of-hatch Ross 708
(Rasheed et al., 2014). Food products contaminated broiler chicks were placed into 16 pens within 2 rooms in
with E. coli continue to be a food safety concern and the Animal Wing on campus at the University of Mary-
cause of economic losses to the poultry industry, espe- land. Within each room, 8 pens were placed with 25
cially as production converts to antibiotic-free programs birds per pen. Each pen was 1.5 m (5 feet) wide by 3 m
(Fancher et al., 2020). (10 feet) long and contained new aspen wood shavings.
E. coli is a Gram-negative bacteria, commonly found The current study was conducted because the birds in a
in intestinal tracts of animals and humans, that can concurrent study became ill and on d 29, a subset of
cause a variety of infections and contribute to the spread culled birds was confirmed to be infected with patho-
of antimicrobial resistance (Rasheed et al., 2014). Chick- genic E. coli, along with Infectious Bronchitis, Entero-
ens can serve as hosts for antimicrobial-resistant E. coli coccus durans, and Enterococcus faecium, by the
because there are multiple routes of contamination at Maryland Department of Agriculture Animal Health
each stage of poultry production. Transmission of anti- Diagnostic Laboratory in Frederick, Maryland. Litter
microbial resistant genes can be attributed to (plasmid- samples were collected on d 53. Composite litter samples
mediated) beta-lactamases, efflux pumps, aminoglyco- were collected from approximately 1 cup of litter from
side phosphorylases, hydrolases, and chloramphenicol 15 locations within each pen (5 locations under the
transacetylase, among others (Dame-Korevaar et al., waterline near the back of the pen, 5 locations near the
2019; Pandey and Cascella, 2022). High levels of antimi- middle of the pen, and 5 locations at the front of the
crobial resistance have been found in day-of-hatch chicks pen), homogenized by hand, and stored at 20°C until
(Braykov et al., 2016), which could originate from the further analysis.
birds’ intestinal microbiota (from vertical transmission)
and from the hatchery environment itself (Osman et al.,
2018). Bacterial horizontal transmission occurs within Isolation of E. coli
and between flocks, which leads to widespread transmis-
In the laboratory, litter samples were thawed to room
sion from the farm to the environment (Dame-
temperature and 3 subsamples from each of the 16 pens
Korevaar et al., 2019). Regardless of the origin, E. coli is
(n = 48 subsamples), ranging from 0.10 to 0.30 g, were
capable of developing resistance through the acquisition
aseptically placed into 15-mL conical tubes with 10 mL
of resistance genes via mutations and horizontal gene
of tryptic soy broth (TSB; BD Difco, Sparks, MD).
transfer (Ievy et al., 2020).
Samples were incubated overnight at 37°C. Following
A single bacterium can quickly transfer an antimicro-
incubation, 100 mL of the TSB solution was streaked
bial-resistant gene and spread resistance to the rest of
onto MacConkey agar plates (BD Difco). The plates
the bacterial colony (Martinez and Baquero, 2000). In
were incubated overnight at 37°C. The next day, 3
the United States, broiler chicken litter is commonly
individual bacterial colonies visually resembling E. coli
reused on-farm for several flocks, so long as the litter is
were selected from each MacConkey agar plate, placed
properly managed to reduce pathogenic bacteria
into TSB, incubated overnight at 37°C, and streaked
(Stenutz et al., 2006). Previous studies have also indi-
onto tryptic soy agar (TSA; BD Difco) plates the next
cated a high prevalence (63−100%) of multi-drug resis-
day. Of the 48 subsamples, a total of 68 E. coli isolates
tant E. coli (Kyakuwaire et al., 2019). While proper
were found.
management of reused litter is not harmful and can be
beneficial, the mismanagement of reused litter could be
a source of not only pathogenic bacteria, but also anti- Antimicrobial Susceptibility
microbial resistance.
The objective of this study was to identify and charac- Antimicrobial susceptibility of E. coli isolates was
terize the antimicrobial resistance profile of E. coli examined using the disc diffusion method, with Escheri-
isolated from litter collected from 16 pens that contained chia coli strain ATCC 25922 as the control. Guidelines
broiler chickens. Relationships between phenotypic set forth by the Clinical and Laboratory Standards Insti-
resistance and virulence genes harbored by E. coli iso- tute (CLSI, 2017) and Bauer, et al. (1966) were used for
lates known to contribute to the prevalence of antimi- susceptibility classification. Briefly, isolates were stored
crobial resistant bacteria were investigated. in a suspension of Luria-Bertani (LB) broth (BD Difco)
and 20% glycerol at 20°C. Isolates were streaked onto
TSA, incubated overnight at 37°C, and individual colo-
MATERIALS AND METHODS nies were selected, and inoculated into 5 mL of Milli-Q
Location water until the bacterial suspension reached an optical
density measured at 600 nm (OD 600) of 0.08 to 0.13
Litter samples were collected at the end of a concur- using a spectrophotometer (NanoPhotometer, Implen,
rent broiler chicken experiment conducted from M€unchen, Germany). The bacterial suspensions were
 POULTRY E. COLI ANTIMICROBIAL RESISTANCE 3
vortexed and streaked onto a Mueller Hinton Agar plate our lab collection (Johnson et al., 2008a; de Oliveira
(BD Difco). Each of the 68 isolates were tested against et al., 2020; Barbieri et al., 2021; Newman et al., 2021),
10 antimicrobials: ampicillin (AMP; 10 mg), azithromy- and the negative controls included sterile water in place
cin (AZM; 15 mg), colistin (CT; 10 mg), imipenem (IPM; of DNA. Amplification parameters of the thermocycler
10 mg), norfloxacin (NOR; 10 mg), streptomycin (STR; (Mastercycler X50, Eppendorf, Hamburg, Germany)
10 mg), sulphonamides (S; 300 mg), trimethoprim/sulfa- included an initial denaturing step at 94°C for 5 min, fol-
methoxazole (SXT; 25 mg), tetracycline (TE; 30 mg), lowed by 30 rounds of 94°C for 30 s, 63°C for 30 s, 68°C
cephalothin (KF; 30 mg) (Oxoid, Basingstoke, UK). for 3 min, then a final extension of 72°C for 10 min, and
After incubation at 37°C for 18 hours, resistance break- a hold at 4°C.
points were determined from the CLSI (2017), except The generated PCR products were subjected to elec-
colistin, for which the guidelines in Bauer et al., (1966) trophoresis, performed in a 2% agarose gel (Agarose LE,
were used. For all antimicrobials, isolates were recorded Lonza, Alpharetta, GA) running at 100 V for 90 min.
as resistant to the antimicrobial if the zone diameter The gel was stained with ethidium bromide (0.25%)
(mm) was at or below the CLSI recommended break- solution for 20 min, visualized using an imager (UVP
point. E. coli isolates were classified as either resistant if BioDock-It2 Imager, Analytik Jena, Jena, Germany),
the zone diameter was less than the breakpoints, or sus- and analyzed for the presence of PCR products of the
ceptible if the zone diameter was greater than the break- appropriate size when compared with the lab control
points. To compare the binomial response of either strains for the targeted genes.
resistant or susceptible, the isolates that exhibited inter-
mediate susceptibility to the antimicrobials were also
categorized as resistant.
16s E. coli Confirmation
Isolates of typical morphology from the MacConkey
agar plates were identified as E. coli and confirmed with
DNA Extraction a 16S rRNA PCR that was performed for each sample
Bacterial DNA was obtained from the whole organ- (Lamprecht et al., 2014). Amplification of the gene tar-
isms using the boil prep method (Barbieri et al., get was carried out as described above.
2013). Briefly, isolates were grown at 37°C overnight
on LB agar (BD Difco). Next, an isolated colony was Antimicrobial and Heavy Metal Resistance
inoculated into 1 mL of LB broth and grown over- Genes
night at 37°C. Cultures were centrifuged at 12,000
RCF for 3 min, the supernatant was discarded, cells DNA samples were amplified using PCR in multiplex
were re-suspended in 200 mL of molecular-grade water, panels to amplify a series of common antimicrobial resis-
and boiled at 100°C (Isotemp, Fisher Scientific, Dubu- tance genes and heavy metal resistance genes harbored
que, IA) for 10 min. After cooling, the suspension was by Enterobacteriaceae species. The genes of interest
centrifuged at 12,000 RCF for 3 min to precipitate cel- were: blaTEM, aac 3VI, tetB, tetA, groEL. aph(3)IA,
lular debris, and 150 mL of the supernatant was trans- dfr17, silP, intl1, pcoD, sull, ISEc12, aadA, aac3-VI,
ferred to a new tube and used as the DNA template and qacEdelta1 (Johnson et al., 2008b; de Oliveira et al.,
for gene amplification. The DNA extracts were stored 2020; Barbieri et al., 2021; Newman et al., 2021).
at 20°C until use.
Serotyping
Polymerase Chain Reaction Amplification The E. coli isolates were tested for their O-antigen
Polymerase chain reaction (PCR) analysis for O-anti- serogroup using PCR (Iguchi et al., 2015). The O-anti-
gen serotyping (Iguchi et al., 2015), characterizing anti- gen serogroups included in the PCR were O1, O2, O8,
microbial resistance and heavy metal genes O15, O18, O25, O26, O29, O30, O55, O75, O78, O84,
(Johnson et al., 2008b), and APEC minimal predictor O86, O8,8 O91, O103, O111, O113, O115, O119, O121,
genes (Johnson et al., 2008a) was carried out using the O12,8 O13,2 O138, O145, O150, O152, O157, O160,
following protocol with minor modifications for anneal- O161, O165, O166, and O183.
ing temperatures of the primers (de Oliveira et al., 2020;
Barbieri et al., 2021; Newman et al., 2021). APEC-Like Minimal Predictors
All PCR reactions were prepared in a total volume of
25 mL for each isolate. Components for the PCR reac- A multiplex PCR was performed with the confirmed
tion consisted of 2.5 mL of 10X PCR buffer, 0.4 mL of E. coli isolates to identify the presence or absence of
10 mM MgCL2, 1 mL of 0.2M dNTP mixture, 2 mL of genes found in APEC isolates (Johnson et al.,
TAQ polymerase (Dream TAQ, ThermoFisher, Wal- 2008a,2008b; Logue et al., 2012). The detection of at
tham, MA), 1.2 mL of primer pool, 2 mL of DNA, and least 3 of the 9 plasmid (cvaC, iroN, ompTp, hlyF, etsB,
15.9 mL of sterile molecular grade water. Positive control iss, aerJ), chromosomal (ireA, papC), or a combination
strains were included in the analysis for the appropriate of the plasmid and chromosomal genes classified the E.
genes of interest from previously characterized strains in coli isolates as APEC-like. The genes used to classify
4 KHONG ET AL.

APEC-like strains cannot definitively conclude that the and the greatest was to cephalothin (54.4%) (Figure 1).
isolated E. coli are truly APEC because they were iso- The percentage of isolates that exhibited resistance to
lated from the litter and not directly isolated from sulphonamides, colistin, ampicillin, tetracycline, and
diseased birds, but they are likely prospects streptomycin were 8.8%, 13.2%, 20.6%, 27.9%, and
(Johnson et al., 2008a). DNA samples were amplified 29.4%, respectively.
using PCR in multiplex panels to amplify a series of 23 Phenotypic multidrug resistance profiles are pre-
virulence-associated genes in APEC. sented in Table 1. A total of 22 (32.4%) isolates were
susceptible to all 10 antimicrobials (Table 1). The
remaining 46 (67.6%) isolates exhibited 21 unique
Statistical Analysis patterns of antimicrobial resistance. The most preva-
To determine if the presence of the virulence genes lent profiles of isolate resistance were to: cephalothin
were more likely to be present in isolates that were resis- (16.2%); cephalothin and streptomycin (5.9%); colis-
tant to antimicrobials, a chi-square contingency analysis tin and cephalothin (4.4%); and tetracycline and
for each pair of antimicrobials and genes was performed streptomycin (4.4%). Multidrug resistance, which is
in JMP Pro (version 14.2, SAS Institute, Inc., Cary, resistance to 3 or more antimicrobials, was found in
NC). Data was considered significant at P ≤ 0.05. 22.1% of the isolates (Table 1). Resistance to 1 anti-
microbial was found in 23.5% of the isolates; 22.1%
were resistant to 2 antimicrobials; 8.8% were resistant
RESULTS to 3 antimicrobials; 5.9% were resistant to 4 antimi-
crobials; 5.9% were resistant to 5 antimicrobials, and
Antimicrobial Susceptibility 1.5% of the isolates were resistant to 6 antimicrobials
The zones of inhibition of the 68 isolates to 10 antimi- (Table 1). Resistance/susceptibility profiles for each
crobial discs in the disc diffusion assays were measured isolate are shown in Table 2.
to determine antimicrobial susceptibility/resistance.
The abbreviations used for each antimicrobial in
Tables 1 and 2 and Figure 1 are as follows: ampicillin O-Antigen Serotyping
(AMP), colistin (CT), tetracycline (TET), sulphona- The PCR-based method was used to determine that a
mides (S3), cephalothin (KF), and streptomycin (S). total of 4 O-serogroups were identified from 20.6%
All isolates were susceptible to azithromycin, trimetho- isolates and of those isolates 57.1% were the serotype
prim/sulfamethoxazole, and norfloxacin, and the cumula- O15, 21.4% were O91, 14.3% were O78, and 7.1% were
tive resistance of isolates to the other 7 antimicrobials is serotype O75 (Table 2).
summarized in Figure 1. Of the isolates that exhibited
resistance, the lowest percentage was to imipenem (1.5%)
PCR Amplification of Heavy Metal Genes and
Antimicrobial Resistance Genes
Table 1. Counts of E. coli isolates (n = 68) sourced from the lit-
ter of broiler chickens raised in 16 pens that were susceptible to all
Antimicrobial and heavy metal resistance gene preva-
antibiotics and unique patterns of resistance to cephalothin (KF), lence are presented in Figure 2. The most prevalent
streptomycin (S), tetracycline (TET), ampicillin (AMP), colistin genes were pcoD at 23.5% and groEL at 20.6%, and tetB
(CT), sulphonamides (S3), and imipenem (IPM). was the least prevalent, found in 2.9% of the isolates. At
Antimicrobial # Isolates
least 1 antimicrobial or heavy metal resistance-associ-
ated gene was present in 69.1% of the isolates. One
AMP 1
KF 11
antimicrobial resistance gene, heavy metal resistance
S 2 gene, or both were present in 35.3% of the isolates, 2
TET 2 genes were detected in 13.2%, 3 genes in 10.3%, 4 genes
AMP KF 2
KF S 4
in 0.0%, 5 genes in 2.9%, 6 genes in 2.9%, and 7 genes in
CT KF 3 4.4% of isolates examined (Table 2).
CT TET 1
TET KF 2
TET S 3
AMP KF S 1 APEC-like Minimal Predictors
AMP CT KF 1
CT TET KF 2 A total of 9 APEC genes were assayed (cvaC, etsB,
TET KF S 2 aerJ, iss, iroN, ompTp, hlyF, ireA, and papC), and iso-
AMP S3 KF S 1
AMP TET KF S 2 lates were considered APEC-like if they possessed 3 or
AMP TET S3 KF 1 more APEC genes (Johnson et al., 2008a). Of the 68 iso-
AMP CT S3 KF S 1 lates, 38.2% were considered APEC-like (Figure 3).
AMP CT TET KF S 1
AMP TET S3 KF S 2 None of the isolates possessed the papC gene. The most
AMP TET IPM S3 KF S 1 prevalent genes were iroN, ompTp, and hlyF; each found
Total Susceptible 22 together in 38.2% of isolates and occurred concurrently
Total 68
in 96.2% of those isolates (Figure 3). Isolates also had
Table 2. The cumulative prevalence and individual isolate presence (black) or absence (white) of antimicrobial resistance O-type gene expression, antimicrobial and heavy
metal resistance genes, and genes indicative of avian pathogenic E. coli (APEC) of E. coli isolates (n = 68) from the litter of broiler chickens raised in 16 pens. Antimicrobials
are abbreviated as AMP (ampicillin), CT (colistin), TET (tetracycline), IMP (imipenem), S3 (sulphonamides), KF (cephalothin), and S (streptomycin) and genes are tetB,
tetA, groEL, aph(3)IA, silP, intl1, pcoD, sull, ISEc12, aadA, aac3-VI, qacEdelta1, cvaC, iroN, ompTp, hlyF, etsB, iss, aerJ, and ireA.

POULTRY E. COLI ANTIMICROBIAL RESISTANCE

(continued on next page)

5
 6
Table 2 (Continued)

KHONG ET AL.
 POULTRY E. COLI ANTIMICROBIAL RESISTANCE 7

Figure 1. Antimicrobial susceptibility prevalence of E. coli isolates (n = 68) from the litter of broiler chickens raised in 16 pens and tested for the
phenotypic resistance (orange) or susceptibility (blue) to ampicillin (AMP), colistin (CT), tetracycline (TET), imipenem (IPM), sulphonamides
(S3), cephalothin (KF), and streptomycin (S). All isolates were susceptible to azithromycin, sulfamethoxazole/trimethoprim, and norfloxacin and
are not shown.

Figure 2. The prevalence (%) of antimicrobial (blaTEM, aac 3VI, tetB, tetA, aph(3)IA, dfr17, aadA, aac3-VI) and heavy metal (silP, pcoD,
sull, qacEdelta1) resistance genes and essential functional genes (groEL, intl1, ISEc12) of E. coli isolates (n = 68) from the litter of broiler chickens
raised in 16 pens. The blaTEM, dfr17, and aac 3VI gene were not present in any of the isolates.

Figure 3. The prevalence (%) of 9 genes (cvaC, iroN, ompTp, hlyF, etsB, iss, aerJ, ireA, and papC) as minimal predictors of avian pathogenic
E. coli (APEC) from isolates (n = 68) sourced from the litter of broiler chickens raised in raised in 16 pens. The presence of at least 3 genes is needed
to be categorized as APEC-like. The papC gene was not present in any of the isolates.
8 KHONG ET AL.

iss, aerJ, etsB, cvaC, and ireA at 33.8, 19.1, 11.8, 7.4, cephalothin, tetracycline, and streptomycin did not
and 1.5%, respectively (Figure 3). have significant associations with APEC genes. How-
ever, there was a greater likelihood that APEC genes
iroN, ompTp, and hlyF were present when isolates were
Antimicrobial Phenotypic Resistance and resistant to ampicillin (P = 0.03).
Genotype
The phenotypic resistance of isolates and the genes DISCUSSION
that they presented were assessed for potential relation-
ships. Of the isolates that were resistant to cephalothin The present study investigated E. coli isolated from
(n = 37 isolates), 85.7% (12/14) were positive for groEL the litter of broiler chickens to characterize antimicrobial
and 100.0% (5/5) were positive for tetA genes. The chi- susceptibility and to determine the relationship between
square analysis showed a significant association between phenotypic resistance and select virulence genes.
cephalothin resistance and the genes groEL (P = Antimicrobial resistance in the poultry industry is of
0.0009) and tetA (P = 0.04). great concern. E. coli can have significant impacts on
Of the isolates resistant to tetracycline (n = 19 consumers when purchasing poultry meat, and on physi-
isolates), 100.0% (5/5) harbored tetA, 57.1% (8/14) cians and patients such as in the treatment of urinary
harbored groEL, 58.3% (7/12) harbored aadA, 58.3% tract infections (Barbieri et al., 2017). Of the 68 isolates
(7/12) harbored aac3-VI, and 66.7% (6/9) habored sampled from the litter of broilers in our study, the
qacEdelta1. The chi-square analysis showed a significant greatest phenotypic resistance was to cephalothin
association between tetracycline resistance and the (54.4%). These results align with those of previous stud-
genes tetA (P = 0.001), groEL (P = 0.01), aadA ies, which have also indicated high prevalence of E. coli
(P = 0.02), aac3-VI (P = 0.02), and qacEdelta1 resistance to cephalothin in poultry. In another study,
(P = 0.01). cloacal swabs from birds with a history of colibacillosis
Of the isolates resistant to streptomycin (n = 20 iso- were used in the analysis of cephalothin resistance of
lates), 56.3% (9/16) presented the pcoD gene, and 30 E. coli isolates from broiler farms in Thailand, with
75.0% (9/12) presented the aadA gene. The chi-square a resistance rate of 73% (Mooljuntee et al., 2010).
analysis showed a significant association between strep- Similarly, a study originating from Korea isolated 591
tomycin resistance and the genes pcoD (P = 0.01) and E. coli isolates from both feces and dust and reported
aadA (P = 0.0004). that the first generation cephalosporins had the highest
Of the isolates resistant to ampicillin (n = 14 isolates), incidences of resistance, ranging from 60 to 71%
42.9% (6/14) presented groEL, 66.7% (4/6) presented (Seo et al., 2019).
aph(3)IA, 75.0% (3/4) presented silP, 37.5% (6/16) Tetracycline has been registered for use in the United
presented pcoD, 72.7% (8/11) presented sull, 58.3% pre- States, China, Poland, United Kingdom, France, Brazil,
sented (7/12) aadA, 66.7% (6/9) presented qacEdelta1, and Spain for therapeutic, metaphylactic/prophylactic,
34.6% (9/26) presented iroN, 34.6% (9/26) presented and growth promotion purposes for over 50 yr
ompTp, and 34.6% (9/26) presented hlyF. The chi- (Barbieri et al., 2015; Roth et al., 2019). Resistance to
square analysis showed a significant association between tetracycline is associated with large plasmids that
ampicillin resistance and the genes groEL (P = 0.02), encode efflux genes which regulate the internal environ-
aph(3)IA (P = 0.01), silP (P = 0.02), sull (P = 0.0001), ment of the Gram-negative bacteria (Soto, 2013). In
aadA (P = 0.002), qacEdelta1 (P = 0.002), iroN E. coli, these large plasmids can also carry other
(P = 0.03), ompT (P = 0.03), and hlyF (P = 0.03). genes such as those responsible for pathogenic factors,
Of the isolates resistant to colistin (n = 9 isolates), antimicrobial resistance, and heavy metal resistance
35.7% (5/14) presented the groEL gene. The chi-square (Diarrassouba et al., 2007). A study that analyzed the
analysis showed a significant association between colis- susceptibility of 144 APEC isolates from cellulitis lesions
tin resistance and the groEL gene (P = 0.01). of broiler chickens found that 69% of isolates exhibited
Of the isolates that exhibited resistance to sulphona- resistance to tetracycline (Barbieri et al., 2013), while
mides (n = 6), 60.0% (3/5) presented for tetA, 35.7% our study indicated that 27.9% of E. coli isolates were
(5/14) presented groEL, 66.7% (4/6) presented aph(3) resistance towards tetracycline. A study by
IA, 75.0% (3/4) presented silP, 25.0% (4/16) presented Smith et al. (2007) analyzed cecal droppings found on
pcoD, 54.5% (6/11) presented sull, 50.0% (6/12) the surface of the litter at 3 untreated commercial broiler
presented aadA, and 66.7% (6/9) presented qacEdelta1 farms at wk 3 and 6. Their study sampled 450 E. coli iso-
gene. The chi-square analysis showed a significant lates and indicated a greater prevalence of tetracycline
association between sulphonamide resistance and the resistance, ranging from 36% at wk 3 to 97% at wk 6
genes tetA (P = 0.004), groEL (P = 0.001), aph(3)IA (Smith et al., 2007). Previous work with E. coli in Brazil
(P = 0.0003), silP (P = 0.002), pcoD (P = 0.02), sull investigated 52 APEC isolates from systemic colibacillo-
(P = 0.0001), aadA (P = 0.0001), and qacEdelta1 sis and observed that 69% of isolates exhibited tetracy-
(P = 0.0001). cline resistance (Barbieri et al., 2015). Our study also
There were no significant associations for genes with indicated moderate resistance to tetracycline, likely
imipenem resistance. Isolates that were resistant to attributed to its long and common use in poultry, which
 POULTRY E. COLI ANTIMICROBIAL RESISTANCE 9
is supported by the finding of tetracycline resistant bac- Kingdom, France, Spain, and Germany (Roth et al.,
teria in birds that were not administered this antimicro- 2019) and were banned between 2015 and 2016 in Brazil
bial (van den Bogaard and Stobberingh 2000; Japan, India, and China (Liu et al., 2016; Schoen-
Agyare, 2019). In general, the lower rates of tetracycline makers, 2020). While colistin is approved for use in the
resistance in the current study compared to the previous United States, it is not approved for commercial poultry
studies could be a result of the absence of tetracycline use. The potential benefits of this ban are significant in
and antimicrobial administration. Further, the use of reducing the use of antimicrobials and decreasing the
tetracycline in the poultry industry has greatly reduced spread of antimicrobial resistance genes; however, it
since 2015 after U.S. FDA implementation of the GFI does not offer a direct solution to the present public
#209 (Singer et al., 2020). Consequently, less antimicro- health concerns caused by antimicrobial resistant bacte-
bial residues would have been shed from the broilers and ria and their genes.
less instances of E. coli resistance to tetracycline would Our study indicated minimal (1.5%) resistance to imi-
be observed. penem, and none of the isolates presented a significant
Streptomycin is approved for use in Brazil and is sel- association with any of the genes of interest. Imipenem
dom used in the United States for the same purposes as resistance is mostly associated with the Gram-negative
listed for tetracycline (Roth et al., 2019). In our study, Enterobacteriaceae Klebsiella pneumoniae (K. pneumo-
27.9% of E. coli isolates exhibited resistance to tetracy- niae) that produces K. pneumoniae carbapenemase
cline, which was lower compared to other work that also (Karlsson et al., 2022). A study by Karlsson et al. (2022)
isolated E. coli from diseased broilers that were not surveyed carbapenemase producing and non-carbapene-
treated with any antimicrobials. According to 2 different mase producing bacteria collected from clinical laborato-
studies in Egypt, there were greater instances of strepto- ries in 8 U.S. states. Of the 419 isolates analyzed, only 23
mycin resistance reported at 74% (Younis et al., 2017) E. coli isolates exhibited carbapenem resistance com-
and 80% resistance (Amer et al., 2018). pared to the 242 K. pneumoniae-resistant isolates
Smith et al. (2007) provided a range of 53 to 100% of E. (Karlsson et al., 2022). It could be that the minimal imi-
coli streptomycin resistance from 3 untreated commer- penem resistance seen in our study is due to the mecha-
cial broiler houses for the United States. In Nigeria, nism of carbapenem resistance, which depends on
Okorafor et al. (2019) observed a range of 10 to 80% of mobile genetic elements derived from K. pneumoniae
streptomycin resistance in broiler chicks from 4 different (Nordmann and Poirel, 2019). Since the birds in our
hatcheries. The results from our study are lower than study were confirmed to be infected with E. coli and not
the findings of these previous studies, which may be K. pneumoniae, a future endeavor would include survey-
attributed to the single (litter) source of our E. coli iso- ing the microbial population of the broiler litter in addi-
lates or that birds were reared in a controlled research tion to testing for antimicrobial resistance.
setting in the current study. Sulphonamide resistance was exhibited in 8.8% of our
The prevalence of E. coli ampicillin resistance in this isolates. This prevalence of resistance is in the lower
study was 20.6% and this is also lower than findings of range of our study. Sulphonamide use in the poultry
previous studies. A study originating from Thailand col- industry is limited due to the high potential for the birds
lected 30 cloacal swabs from broilers on commercial to develop toxic side effects (Landoni and Albarel-
farms and found that 100% of the E. coli isolates exhib- los, 2015). A previous study by Furtula et al. (2010)
ited ampicillin resistance (Mooljuntee et al., 2010). assessed the antimicrobial resistance of E. coli in both
From the studies mentioned previously by commercial and controlled broiler feeding trials. It was
Younis et al. (2017) and Amer et al. (2018), 47 and 80% suggested that E. coli resistance to sulphonamides was
of E. coli isolates exhibited ampicillin resistance, respec- present in the chicks since hatch because of the existing
tively. In Nigeria, Okorafor et al. (2019) observed that sulphonamide resistant E. coli found in the birds of the
80 to 100% of the isolates from broiler chicks exhibited control group at d 36 (Furtula et al., 2010). Since the
ampicillin resistance. Ampicillin is approved for use in isolates resistant to sulphonamides originated from birds
Brazil, China, Germany, and France (Roth et al., 2019), that were housed in pens in the same facility, it is possi-
but is not approved for use in the United States for any ble that as chicks, the birds used in our study were from
purpose in poultry and livestock, which may explain our a breeder flock where sulphonamide resistance existed.
study’s lower prevalence of ampicillin resistance. This study also sought to identify genes indicative of
Interestingly, 13.2% of E. coli isolates were resistant avian pathogenic E. coli. As described by
to colistin in our study. Resistance to colistin is concern- Johnson et al. (2008a), there are genes that can be indic-
ing because it is a last-resort antimicrobial used to treat ative of APEC, and isolates must have at least 3 of the
bacterial infections in humans. The plasmid-mediated following 9 genes to be considered APEC-like: cvaC,
mobile colistin resistance (mcr) gene encodes colistin iroN, ompTp, hlyF, etsB, iss, aerJ, ireA, or papC. As a
resistance (Barbieri et al., 2017), and it is mostly found plasmid of interest, ColV is thought to assist APEC
in E. coli isolated from swine, bovine, poultry, and their strains in infection (Barbieri et al., 2013). The ColV
related products (Valiakos and Kapna, 2021). The use plasmid is associated with genes such as cvi/cva, iroN,
of colistin in poultry was approved in many countries by iss, iucD, sitD, traT, and tsh (Barbieri et al., 2015). Our
their respective national regulatory authorities, includ- study found that the genes iroN, ompTp, and hlyF were
ing the United States, Brazil, China, Poland, the United the most prevalent (38.2%) among isolates and that all
10 KHONG ET AL.

3 genes were present together 96.2% of the time. DISCLOSURES

Although our study did not assess plasmid genes as in
previous studies (Peigne et al., 2009; Mahjoub- The authors declare no conflicts of interest.
Messai et al., 2011), it is possible that the isolates that
contained the genes iroN, ompTp, hlyF, and iss could
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