Journal Pre-proof

Antimicrobial resistance profile of Escherichia coli isolated from

poultry litter

M.J. Khong , A.M. Snyder , A.K. Magnaterra , M.M. Young ,

N.L. Barbieri , S.L. Weimer

PII: S0032-5791(22)00599-5
DOI: https://doi.org/10.1016/j.psj.2022.102305
Reference: PSJ 102305

To appear in: Poultry Science

Received date: 15 July 2022

Accepted date: 27 October 2022

Please cite this article as: M.J. Khong , A.M. Snyder , A.K. Magnaterra , M.M. Young ,
N.L. Barbieri , S.L. Weimer , Antimicrobial resistance profile of Escherichia coli isolated from
poultry litter, Poultry Science (2022), doi: https://doi.org/10.1016/j.psj.2022.102305

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 Poultry E. coli Antimicrobial Resistance

Antimicrobial resistance profile of Escherichia coli isolated from poultry litter

M. J. Khong1, A. M. Snyder1, A. K. Magnaterra1, M. M, Young2, N. L. Barbieri2, and S. L.

Weimer1,3*

1
Department of Animal and Avian Sciences, University of Maryland, College Park, MD, 20742
2
Department of Population Health, University of Georgia College of Veterinary Medicine,

Athens, GA, 30602

3
Department of Poultry Science, University of Arkansas, Fayetteville AR, 72701

The authors declare no conflicts of interest.

*Corresponding author: Shawna Weimer, Assistant Professor, University of Arkansas, 1260 W

Maple St., Fayetteville, AR 72701, [email protected]

ABSTRACT Antimicrobial resistance is a threat to animal and human health. As a commensal

and zoonotic bacterium, Escherichia coli has the potential to be a pathogenic source of

antimicrobial resistance. The purpose of this study aimed to investigate the antimicrobial

resistance profile of E. coli isolated from litter collected from pens in a broiler chicken

experiment. E. coli was isolated from the litter samples (n = 68 isolates) of 16 pens housing

broilers to d 53 of age. Resistance to 10 antimicrobials was observed by disc diffusion. The

presence of 23 antimicrobial and heavy metal resistance genes, O serogroups, and avian

pathogenic E. coli (APEC-like) minimal predictor genes were identified through PCR. E. coli

isolates presented the greatest resistance to cephalothin (54.4%), tetracycline (27.9%),

streptomycin (29.4%), ampicillin (20.6%), colistin (13.2%), sulphonamides (8.8%), and

 Poultry E. coli Antimicrobial Resistance

imipenem (1.5%). Multidrug resistance to at least 3 antimicrobials was observed in 22.1% of

isolates. The identified O-types of the E. coli isolates were O15, O75, O78, and O91. There was

a greater likelihood that the genes groEL, aph(3)IA, silP, sull, aadA, qacEdelta1, iroN, ompTp,

and hlyF were present in isolates that exhibited ampicillin resistance (P ≤ 0.05). There was a

greater likelihood that the groEL gene was present in isolates resistant to either ampicillin,

colistin, tetracycline, sulphonamides, or cephalothin (P ≤ 0.05). Further characterizing E. coli

antimicrobial resistance is essential and aids in developing effective solutions, thereby furthering

the One Health objective.

Keywords: Escherichia coli, APEC, poultry litter, antimicrobial resistance, disc diffusion

INTRODUCTION

Antimicrobial resistance is one of the most important global health issues because it

affects human and animal populations (World Health Organization, 2019). The U.S. Food and

Drug Administration reported that 2.8 million people in the United States contracted an

antimicrobial-resistant infection in 2019, leading to over 35,000 deaths (United States Food and

Drug Administration, 2019). The One Health concept arises from the intersections of animals,

humans, and the environment and aims to attain optimal public health by preventing and

controlling zoonotic diseases (One Health Initiative Task Force, 2012; Bidaisee and Macpherson,

2014). Infections caused by antimicrobial-resistant bacteria can exacerbate existing

complications in treatment and increase mortality in humans and animals (Munita and Arias,

2016).
 Poultry E. coli Antimicrobial Resistance

Commonly found as a commensal (non-pathogenic) organism, E. coli can become

pathogenic by acquiring virulence factors on plasmids or other mobile genetic elements via

horizontal gene transfer, thus enabling certain strains of E. coli to cause intestinal or

extraintestinal disease (Kaper et al., 2004; de Oliveira et al., 2020). A pathotype of concern in the

poultry industry is avian pathogenic E. coli (APEC), which is the etiological agent of

colibacillosis and can manifest as infections such as airsacculitis, polyserositis, and septicemia

(Dho-Moulin and Fairbrother, 1999). Colibacillosis negatively impacts the health and welfare of

poultry and incidence can be related to the quality of feed, water and litter, antimicrobial use and

stewardship, and management practices (Nolan et al., 2013). Morbidity from E. coli infections

can increase the use of antimicrobials for therapeutic treatment, resulting in significant economic

losses for poultry producers. Mounting evidence suggests that APEC-contaminated poultry is a

source of extraintestinal pathogenic E. coli causing human disease (Rodriguez-Siek et al., 2005;

Vincent et al., 2010; Manges and Johnson, 2012; Manges, 2016). In a study comparing the

incidences of antimicrobial resistance across food products (such as vegetable salads and raw

meats), raw chicken was reported to have the highest E. coli incidence (23.3%) of antimicrobial

resistance (Rasheed et al., 2014). Food products contaminated with E. coli continue to be a food

safety concern and cause of economic losses to the poultry industry, especially as production

converts to antibiotic-free programs (Fancher et al., 2020).

E. coli is a Gram-negative bacteria, commonly found in intestinal tracts of animals and

humans, that can cause a variety of infections and contribute to the spread of antimicrobial

resistance (Rasheed et al., 2014). Chickens can serve as hosts for antimicrobial-resistant E. coli

because there are multiple routes of contamination at each stage of poultry production.
 Poultry E. coli Antimicrobial Resistance

Transmission of antimicrobial resistant genes can be attributed to (plasmid-mediated) beta-

lactamases, efflux pumps, aminoglycoside phosphorylases, hydrolases, and chloramphenicol

transacetylase, among others (Dame-Korevaar et al., 2019; Pandey and Cascella, 2022). High

levels of antimicrobial resistance have been found in day-of-hatch chicks (Braykov et al., 2016),

which could originate from the birds’ intestinal microbiota (from vertical transmission) and from

the hatchery environment itself (Osman et al., 2018). Bacterial horizontal transmission occurs

within and between flocks, which leads to widespread transmission from the farm to the

environment (Dame-Korevaar et al., 2019). Regardless of the origin, E. coli is capable of

developing resistance through the acquisition of resistance genes via mutations and horizontal

gene transfer (Ievy et al., 2020).

A single bacterium can quickly transfer an antimicrobial-resistant gene and spread

resistance to the rest of the bacterial colony (Martinez and Baquero, 2000). In the U.S., broiler

chicken litter is commonly reused on-farm for several flocks, so long as the litter is properly

managed to reduce pathogenic bacteria (Stenutz et al., 2006). Previous studies have also

indicated a high prevalence (63%-100%) of multi-drug resistant E. coli (Kyakuwaire et al.,

2019). The mismanagement of reused litter could be a source of not only pathogenic bacteria, but

also antimicrobial resistance.

The objective of this study was to identify and characterize the antimicrobial resistance

profile of E. coli isolated from litter collected from 16 pens that contained broiler chickens.

Relationships between phenotypic resistance and virulence genes harbored by E. coli isolates

known to contribute to the prevalence of antimicrobial resistant bacteria were investigated.

 Poultry E. coli Antimicrobial Resistance

MATERIALS AND METHODS

Location

Litter samples were collected at the end of a concurrent broiler chicken experiment

conducted from September to November 2020. The birds were not vaccinated or treated with any

antimicrobials. All animal procedures were approved by the University of Maryland Institutional

Care and Use Committee (protocol #R-JUL-20-35). Briefly, 400 day-of-hatch Ross 708 broiler

chicks were placed into 16 pens within 2 rooms in the Animal Wing on campus at the University

of Maryland. Within each room, 8 pens were placed with 25 birds per pen. Each pen was 1.5 m

(5 feet) wide by 3 m (10 feet) long and contained new aspen wood shavings. The current study

was conducted because the birds in a concurrent study became ill and on d 29, a subset of culled

birds was confirmed to be infected with pathogenic E. coli, along with Infectious Bronchitis,

Enterococcus durans, and Enterococcus faecium, by the Maryland Department of Agriculture

Animal Health Diagnostic Laboratory in Frederick, Maryland. Litter samples were collected on d

53. Composite litter samples were collected from approximately 1 cup of litter from 15 locations

within each pen (5 locations under the waterline near the back of the pen, 5 locations near the

middle of the pen, and 5 locations at the front of the pen), homogenized by hand, and stored at -

20ºC until further analysis.

Isolation of E. coli

In the laboratory, litter samples were thawed to room temperature and 3 subsamples from

each of the 16 pens (n = 48 subsamples), ranging from 0.10-0.30 g, were aseptically placed into

15-mL conical tubes with 10 mL of tryptic soy broth (TSB) (BD Difco™, Sparks, MD). Samples
 Poultry E. coli Antimicrobial Resistance

were incubated overnight at 37ºC. Following incubation, 100 µL of the TSB solution was

streaked onto MacConkey agar plates (BD Difco™, Sparks, MD). The plates were incubated

overnight at 37ºC. The next day, 3 individual bacterial colonies visually resembling E. coli were

selected from each MacConkey agar plate, placed into TSB, incubated overnight at 37ºC, and

streaked onto tryptic soy agar (TSA) (BD Difco™, Sparks, MD) plates the next day. Of the 48

subsamples, a total of 68 E. coli isolates were found.

Antimicrobial Susceptibility

Antimicrobial susceptibility of E. coli isolates was examined using the disc diffusion

method, with Escherichia coli strain ATCC 25922 as the control. Guidelines set forth by the

Clinical and Laboratory Standards Institute (CLSI, 2017) and Bauer, et al. (1966) were used for

susceptibility classification. Briefly, isolates were stored in a suspension of Luria-Bertani (LB)

broth (BD Difco™, Sparks, MD) and 20% glycerol at -20ºC. Isolates were streaked onto TSA,

incubated overnight at 37ºC, and individual colonies were selected, and inoculated into 5 mL of

Milli-Q water until the bacterial suspension reached an optical density measured at 600 nm (OD

600) of 0.08-0.13 using a spectrophotometer (NanoPhotometer, Implen, München, Germany).

The bacterial suspensions were vortexed and streaked onto a Mueller Hinton Agar plate (BD

Difco™, Sparks, MD). Each of the 68 isolates were tested against 10 antimicrobials: ampicillin

(AMP; 10 µg), azithromycin (AZM; 15 µg), colistin (CT; 10 µg), imipenem (IPM; 10 µg),

norfloxacin (NOR; 10 µg), streptomycin (STR; 10 µg), sulphonamides (S; 300 µg),

trimethoprim/sulfamethoxazole (SXT; 25 µg), tetracycline (TE; 30 µg), cephalothin (KF; 30 µg)

(Oxoid, Basingstoke, UK). After incubation at 37ºC for 18 hours, resistance breakpoints were

determined from the CLSI (2017), except colistin, for which the guidelines in Bauer et al.,
 Poultry E. coli Antimicrobial Resistance

(1966) were used. For all antimicrobials, isolates were recorded as resistant to the antimicrobial

if the zone diameter (mm) was at or below the CLSI recommended breakpoint. E. coli isolates

were classified as either resistant if the zone diameter was less than the breakpoints, or

susceptible if the zone diameter was greater than the breakpoints. To compare the binomial

response of either resistant or susceptible, the isolates that exhibited intermediate susceptibility

to the antimicrobials were also categorized as resistant.

DNA Extraction

Bacterial DNA was obtained from the whole organisms using the boil prep method

(Barbieri et al., 2013). Briefly, isolates were grown at 37ºC overnight on LB agar (BD Difco™,

Sparks, MD). Next, an isolated colony was inoculated into 1 ml of LB broth and grown

overnight at 37ºC. Cultures were centrifuged at 12,000 RCF for 3 minutes, the supernatant was

discarded, cells were re-suspended in 200 μL of molecular-grade water, and boiled at 100ºC

(Isotemp, Fisher Scientific, Dubuque, IA) for 10 minutes. After cooling, the suspension was

centrifuged at 12,000 RCF for 3 minutes to precipitate cellular debris, and 150 μL of the

supernatant was transferred to a new tube and used as the DNA template for gene amplification.

The DNA extracts were stored at −20ºC until use.

Polymerase Chain Reaction (PCR) Amplification

PCR analysis for O-antigen serotyping (Iguchi et al., 2015), characterizing antimicrobial

resistance and heavy metal genes (Johnson et al., 2008b), and APEC minimal predictor genes

(Johnson et al., 2008a) was carried out using the following protocol with minor modifications for
 Poultry E. coli Antimicrobial Resistance

annealing temperatures of the primers (de Oliveira et al., 2020; Barbieri et al., 2021; Newman et

al., 2021).

All PCR reactions were prepared in a total volume of 25 µL for each isolate. Components

for the PCR reaction consisted of 2.5 µL of 10X PCR buffer, 0.4 µL of 10 mM MgCL2, 1 µL of

0.2M dNTP mixture, 2 µL of TAQ polymerase (Dream TAQ, ThermoFisher, Waltham, MA), 1.2

µL of primer pool, 2 µL of DNA, and 15.9 µL of sterile molecular grade water. Positive control

strains were included in the analysis for the appropriate genes of interest from previously

characterized strains in our lab collection (Johnson et al., 2008a; de Oliveira et al., 2020; Barbieri

et al., 2021; Newman et al., 2021), and the negative controls included sterile water in place of

DNA. Amplification parameters of the thermocycler (Mastercycler X50, Eppendorf, Hamburg,

Germany) included an initial denaturing step at 94ºC for 5 minutes, followed by 30 rounds of

94ºC for 30 seconds, 63ºC for 30 seconds, 68ºC for 3 minutes, then a final extension of 72ºC for

10 minutes, and a hold at 4°C.

The generated PCR products were subjected to electrophoresis, performed in a 2%

agarose gel (Agarose LE, Lonza, Alpharetta, GA) running at 100 V for 90 minutes. The gel was

stained with ethidium bromide (0.25%) solution for 20 minutes, visualized using an imager

(UVP BioDock-It2 Imager, Analytik Jena, Jena, Germany), and analyzed for the presence of

PCR products of the appropriate size when compared with the lab control strains for the targeted

genes.

16s E. coli Confirmation

 Poultry E. coli Antimicrobial Resistance

Isolates of typical morphology from the MacConkey agar plates were identified as E. coli

and confirmed with a 16S rRNA PCR that was performed for each sample (Lamprecht et al.,

2014). Amplification of the gene target was carried out as described above.

Antimicrobial and Heavy Metal Resistance Genes

DNA samples were amplified using PCR in multiplex panels to amplify a series of

common antimicrobial resistance genes and heavy metal resistance genes harbored by

Enterobacteriaceae species. The genes of interest were: blaTEM, aac 3VI, tetB, tetA, groEL.

aph(3)IA, dfr17, silP, intl1, pcoD, sull, ISEc12, aadA, aac3-VI, and qacEdelta1 (Johnson et al.,

2008b; de Oliveira et al., 2020; Barbieri et al., 2021; Newman et al., 2021).

Serotyping

The E. coli isolates were tested for their O-antigen serogroup using PCR (Iguchi et al.,

2015). The O-antigen serogroups included in the PCR were O1; O2; O8; O15; O18; O25; O26;

O29; O30; O55; O75; O78; O84; O86; O88; O91; O103; O111; O113; O115; O119; O121;

O128; O132; O138; O145; O150; O152; O157; O160; O161; O165; O166; O183.

APEC-like Minimal Predictors

A multiplex PCR was performed with the confirmed E. coli isolates to identify the

presence or absence of genes found in APEC isolates (Johnson et al., 2008a, 2008b; Logue et al.,

2012). The detection of at least 3 of the 9 plasmid (cvaC, iroN, ompTp, hlyF, etsB, iss, aerJ),

chromosomal (ireA, papC), or a combination of the plasmid and chromosomal genes classified

the E. coli isolates as APEC-like. The genes used to classify APEC-like strains cannot
 Poultry E. coli Antimicrobial Resistance

definitively conclude that the isolated E. coli are truly APEC because they were isolated from the

litter and not directly isolated from diseased birds, but they are likely prospects (Johnson et al.,

2008a). DNA samples were amplified using PCR in multiplex panels to amplify a series of 23

virulence-associated genes in APEC.

Statistical Analysis

To determine if the presence of the virulence genes were more likely to be present in

isolates that were resistant to antimicrobials, a chi-square contingency analysis for each pair of

antimicrobials and genes was performed in JMP Pro (version 14.2, SAS Institute, Inc., Cary,

NC). Data was considered significant at P ≤ 0.05.

RESULTS

Antimicrobial Susceptibility

The zones of inhibition of the 68 isolates to 10 antimicrobial discs in the disc diffusion

assays were measured to determine antimicrobial susceptibility/resistance. The abbreviations

used for each antimicrobial in Tables 1 and 2 and Figure 1 are as follows: ampicillin (AMP),

colistin (CT), tetracycline (TET), sulphonamides (S3), cephalothin (KF), and streptomycin (S).

All isolates were susceptible to azithromycin, trimethoprim/sulfamethoxazole, and

norfloxacin, and the cumulative resistance of isolates to the other 7 antimicrobials is summarized

in Figure 1. Of the isolates that exhibited resistance, the lowest percentage was to imipenem

(1.5%) and the greatest was to cephalothin (54.4%) (Figure 1). The percentage of isolates that

exhibited resistance to sulphonamides, colistin, ampicillin, tetracycline, and streptomycin was

8.8%, 13.2%, 20.6%, 27.9%, and 29.4%, respectively.

 Poultry E. coli Antimicrobial Resistance

Phenotypic multidrug resistance profiles are presented 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 patterns of antimicrobial resistance. The most prevalent profiles of isolate

resistance were to: cephalothin (16.2%); cephalothin and streptomycin (5.9%); colistin and

cephalothin (4.4%); and tetracycline and streptomycin (4.4%). Multidrug resistance, which is

resistance to 3 or more antimicrobials, was found in 22.1% of the isolates (Table 1). Resistance

to 1 antimicrobial was found in 23.5% of the isolates; 22.1% were resistant to 2 antimicrobials;

8.8% were resistant to 3 antimicrobials; 5.9% were resistant to 4 antimicrobials; 5.9% were

resistant to 5 antimicrobials, and 1.5% of the isolates were resistant to 6 antimicrobials (Table

1). Resistance/susceptibility profiles for each isolate are shown in Table 2.

O-Antigen Serotyping

The PCR-based method was used to determine that a total of 4 O-serogroups were

identified from 20.6% isolates and of those isolates 57.1% were the serotype O15, 21.4% were

O91, 14.3% were O78, and 7.1% were serotype O75 (Table 2).

PCR Amplification of Heavy Metal Genes and Antimicrobial Resistance Genes

Antimicrobial and heavy metal resistance gene prevalence are presented in Figure 2. The

most prevalent genes were pcoD at 23.5% and groEL at 20.6%, and tetB was the least prevalent,

found in 2.9% of the isolates. At least 1 antimicrobial or heavy metal resistance-associated gene

was present in 69.1% of the isolates. One antimicrobial resistance gene or heavy metal resistance

gene or both were present in 35.3% of the isolates, 2 genes were detected in 13.2%, 3 genes in
 Poultry E. coli Antimicrobial Resistance

10.3%, 4 genes in 0.0%, 5 genes in 2.9%; 6 genes in 2.9%, and 7 genes in 4.4% of isolates

examined (Table 2).

APEC-like Minimal Predictors

A total of 9 APEC genes were assayed (cvaC, etsB, aerJ, iss, iroN, ompTp, hlyF, ireA,

and papC), and isolates were considered APEC-like if they possessed 3 or more APEC genes

(Johnson et al., 2008a). Of the 68 isolates, 38.2% were considered APEC-like (Figure 3). None

of the isolates possessed the papC gene. The most prevalent genes were iroN, ompTp, and hlyF;

each found together in 38.2% of isolates and occurred concurrently in 96.2% of those isolates

(Figure 3). Isolates also had iss, aerJ, etsB, cvaC, and ireA at 33.8%, 19.1%, 11.8%, 7.4%, and

1.5%, respectively (Figure 3).

Antimicrobial Phenotypic Resistance and Genotype

The phenotypic resistance of isolates and the genes that they presented were assessed for

potential relationships. Of the isolates that were resistant to cephalothin (n = 37 isolates), 85.7%

(12/14) were positive for groEL and 100.0% (5/5) were positive for tetA genes. The chi-square

analysis showed a significant association between cephalothin resistance and the genes groEL (P

= 0.0009) and tetA (P = 0.04).

Of the isolates resistant to tetracycline (n = 19 isolates), 100.0% (5/5) harbored tetA,

57.1% (8/14) harbored groEL, 58.3% (7/12) harbored aadA, 58.3% (7/12) harbored aac3-VI, and

66.7% (6/9) habored qacEdelta1. The chi-square analysis showed a significant association

between tetracycline resistance and the genes tetA (P = 0.001), groEL (P = 0.01), aadA (P =

0.02), aac3-VI (P = 0.02), and qacEdelta1 (P = 0.01).

 Poultry E. coli Antimicrobial Resistance

Of the isolates resistant to streptomycin (n = 20 isolates), 56.3% (9/16) presented the

pcoD gene, and 75.0% (9/12) presented the aadA gene. The chi-square analysis showed a

significant association between streptomycin resistance and the genes pcoD (P = 0.01) and aadA

(P = 0.0004).

Of the isolates resistant to ampicillin (n = 14 isolates), 42.9% (6/14) presented groEL,

66.7% (4/6) presented aph(3)IA, 75.0% (3/4) presented silP, 37.5% (6/16) presented pcoD,

72.7% (8/11) presented sull, 58.3% presented (7/12) aadA, 66.7% (6/9) presented qacEdelta1,

34.6% (9/26) presented iroN, 34.6% (9/26) presented ompTp, and 34.6% (9/26) presented hlyF.

The chi-square analysis showed a significant association between ampicillin resistance and the

genes groEL (P = 0.02), aph(3)IA (P = 0.01), silP (P = 0.02), sull (P = 0.0001), aadA (P =

0.002), qacEdelta1 (P = 0.002), iroN (P = 0.03), ompT (P = 0.03), and hlyF (P = 0.03).

Of the isolates resistant to colistin (n = 9 isolates), 35.7% (5/14) presented the groEL

gene. The chi-square analysis showed a significant association between colistin resistance and

the groEL gene (P = 0.01).

Of the isolates that exhibited resistance to sulphonamides (n = 6), 60.0% (3/5) presented

for tetA, 35.7% (5/14) presented groEL, 66.7% (4/6) presented aph(3)IA, 75.0% (3/4) presented

silP, 25.0% (4/16) presented pcoD, 54.5% (6/11) presented sull, 50.0% (6/12) presented aadA,

and 66.7% (6/9) presented qacEdelta1 gene. The chi-square analysis showed a significant

association between sulphonamide resistance and the genes tetA (P = 0.004), groEL (P = 0.001),

aph(3)IA (P = 0.0003), silP (P = 0.002), pcoD (P = 0.02), sull (P = 0.0001), aadA (P = 0.0001),

and qacEdelta1 (P = 0.0001).

There were no significant associations for genes with imipenem resistance. Isolates that

were resistant to cephalothin, tetracycline, and streptomycin did not have significant associations
 Poultry E. coli Antimicrobial Resistance

with APEC genes. However, there was a greater likelihood that APEC genes iroN, ompTp, and

hlyF were present when isolates were resistant to ampicillin (P = 0.03).

Discussion

The present study investigated E. coli isolated from the litter of broiler chickens to

characterize antimicrobial susceptibility and to determine the relationship between phenotypic

resistance and select virulence genes.

Antimicrobial resistance in the poultry industry is of great concern. E. coli can have

significant impacts on consumers when purchasing poultry meat, and on physicians and patients

such as in the treatment of urinary tract infections (Barbieri et al., 2017). Of the 68 isolates

sampled from the litter of broilers in our study, the greatest phenotypic resistance was to

cephalothin (54.4%). These results align with those of previous studies, which have also

indicated high prevalence of E. coli resistance to cephalothin in poultry. In another study, cloacal

swabs from birds with a history of colibacillosis were used in the analysis of cephalothin

resistance of 30 E. coli isolates from broiler farms in Thailand, with a resistance rate of 73%

(Mooljuntee et al., 2010). Similarly, a study originating from Korea isolated 591 E. coli isolates

from both feces and dust and reported that the first generation cephalosporins had the highest

incidences of resistance, ranging from 60% to 71% (Seo et al., 2019).

Tetracycline has been registered for use in the United States, China, Poland, United

Kingdom, France, Brazil, and Spain for therapeutic, metaphylactic/prophylactic, and growth

promotion purposes for over 50 years (Barbieri et al., 2015; Roth et al., 2019). Resistance to
 Poultry E. coli Antimicrobial Resistance

tetracycline is associated with large plasmids that encode efflux genes which regulate the internal

environment of the Gram-negative bacteria (Soto, 2013). In E. coli, these large plasmids can also

carry other genes such as those responsible for pathogenic factors, antimicrobial resistance, and

heavy metal resistance (Diarrassouba et al., 2007). A study that analyzed the susceptibility of

144 APEC isolates from cellulitis lesions of broiler chickens found that 69% of isolates exhibited

resistance to tetracycline (Barbieri et al., 2013), while our study indicated that 27.9% of E. coli

isolates were resistance towards tetracycline. A study by Smith et al. (2007) analyzed cecal

droppings found on the surface of the litter at 3 untreated commercial broiler farms at weeks 3

and 6. Their study sampled 450 E. coli isolates and indicated a greater prevalence of tetracycline

resistance, ranging from 36% at week 3 to 97% at week 6 (Smith et al., 2007). Previous work

with E. coli in Brazil investigated 52 APEC isolates from systemic colibacillosis and observed

that 69% of isolates exhibited tetracycline resistance (Barbieri et al., 2015). Our study also

indicated moderate resistance to tetracycline, likely attributed to its long and common use in

poultry, which is supported by the finding of tetracycline resistant bacteria in birds that were not

administered this antimicrobial (van den Bogaard and Stobberingh 2000; Agyare et al., 2019). In

general, the lower rates of tetracycline resistance in the current study compared to the previous

studies could be a result of the absence of tetracycline and antimicrobial administration. Further,

the use of tetracycline in the poultry industry has greatly reduced since 2015 after U.S. FDA

implementation of the GFI #209 (Singer et al., 2020). Consequently, less antimicrobial residues

would have been shed from the broilers and less instances of E. coli resistance to tetracycline

would be observed.
 Poultry E. coli Antimicrobial Resistance

Streptomycin is approved for use in Brazil and is seldom used in the United States for the

same purposes as listed for tetracycline (Roth et al., 2019). In our study, 27.9% of E. coli isolates

exhibited resistance to tetracycline, which was lower compared to other work that also isolated

E. coli from diseased broilers that were not treated with any antimicrobials. According to 2

different studies in Egypt, there were greater instances of streptomycin resistance reported at

74% (Younis et al., 2017) and 80% resistance (Amer et al., 2018). Smith et al. (2007) provided a

range of 53% to 100% of E. coli streptomycin resistance from 3 untreated commercial broiler

houses for the United States. In Nigeria, Okorafor et al. (2019) observed a range of 10% to 80%

of streptomycin resistance in broiler chicks from 4 different hatcheries. The results from our

study are lower than the findings of these previous studies, which may be attributed to the single

(litter) source of our E. coli isolates or that birds our birds were reared in a controlled research

setting.

The prevalence of E. coli ampicillin resistance in this study was 20.6% and this is also

lower than findings of previous studies. A study originating from Thailand collected 30 cloacal

swabs from broilers on commercial farms and found that 100% of the E. coli isolates exhibited

ampicillin resistance (Mooljuntee et al., 2010). From the studies mentioned previously by Younis

et al. (2017) and Amer et al. (2018), 47% and 80% of E. coli isolates exhibited ampicillin

resistance, respectively. In Nigeria, Okorafor et al. (2019) observed that 80% to 100% of the

isolates from broiler chicks exhibited ampicillin resistance. Ampicillin is approved for use in

Brazil, China, Germany, and France (Roth et al., 2019), but is not approved for use in the United

States for any purpose in poultry and livestock, which may explain our study’s lower prevalence

of ampicillin resistance.
 Poultry E. coli Antimicrobial Resistance

Interestingly, 13.2% of E. coli isolates were resistant to colistin in our study. Resistance

to colistin is concerning because it is a last-resort antimicrobial used to treat bacterial infections

in humans. The plasmid-mediated mobile colistin resistance (mcr) gene encodes colistin

resistance (Barbieri et al., 2017), and it is mostly found in E. coli isolated from swine, bovine,

poultry, and their related products (Valiakos and Kapna, 2021). The use of colistin in poultry was

approved in many countries by their respective national regulatory authorities, including the

United States, Brazil, China, Poland, the United Kingdom, France, Spain, and Germany (Roth et

al., 2019) and were banned between 2015 and 2016 in Brazil Japan, India, and China (Liu et al.,

2016; Schoenmakers, 2020). While colistin is approved for use in the U.S., it is not approved for

commercial poultry use. Tthe potential benefits of this ban are significant in reducing the use of

antimicrobials and decreasing the spread of antimicrobial resistance genes; however, it does not

offer a direct solution to the present public health concerns caused by antimicrobial resistant

bacteria and their genes.

Our study indicated minimal (1.5%) resistance to imipenem, and none of the isolates

presented a significant association with any of the genes of interest. Imipenem resistance is

mostly associated with the Gram-negative Enterobacteriaceae Klebsiella pneumoniae (K.

pneumoniae) that produces K. pneumoniae carbapenemase (Karlsson et al., 2022). A study by

Karlsson et al. (2022) surveyed carbapenemase producing and non-carbapenemase producing

bacteria collected from clinical laboratories in 8 U.S. states. Of the 419 isolates analyzed, only

23 E. coli isolates exhibited carbapenem resistance compared to the 242 K. pneumoniae isolates

(Karlsson et al., 2022). It could be that the minimal imipenem resistance seen in our study is due

to the mechanism of carbapenem resistance, which depends on mobile genetic elements derived
 Poultry E. coli Antimicrobial Resistance

from K. pneumoniae (Nordmann and Poirel, 2019). Since the birds in our study were confirmed

to be infected with E. coli and not K. pneumoniae a future endeavor would include surveying the

microbial population of the broiler litter in addition to testing for antimicrobial resistance.

Sulphonamide resistance was exhibited in 8.8% of our isolates. This prevalence of

resistance is in the lower range of our study. Sulphonamide use in the poultry industry is limited

due to the high potential for the birds to develop toxic side effects (Landoni and Albarellos,

2015). A previous study by Furtula et al. (2010) assessed the antimicrobial resistance of E. coli

in both commercial and controlled broiler feeding trials. It was suggested that E. coli resistance

to sulphonamides was present in the chicks since hatch because of the existing sulphonamide

resistant E. coli found in the birds of the control group at d 36 (Furtula et al., 2010). Since the

isolates resistant to sulphonamides originated from birds that were housed in pens in the same

facility, it is possible that as chicks, the birds used in our study were from a breeder flock where

sulphonamide resistance existed. Through coprophagic habits of broilers, and the possible

elimination of the antimicrobial through excrements, the cycle of sulphonamide resistance

continues.

This study also sought to identify genes indicative of avian pathogenic E. coli. As

described by Johnson et al. (2008a), there are genes that can be indicative of APEC, and isolates

must have at least 3 of the following 9 genes to be considered APEC-like: cvaC, iroN, ompTp,

hlyF, etsB, iss, aerJ, ireA, or papC. As a plasmid of interest, ColV is thought to assist APEC

strains in infection (Barbieri et al., 2013). The ColV plasmid is associated with genes such as

cvi/cva, iroN, iss, iucD, sitD, traT, and tsh (Barbieri et al., 2015). Our study found that the genes

iroN, ompTp, and hlyF were the most prevalent (38.2%) among isolates and that all 3 genes were

present together 96.2% of the time. Although our study did not assess plasmid genes as in
 Poultry E. coli Antimicrobial Resistance

previous studies (Peigne et al., 2009; Mahjoub-Messai et al., 2011), it is possible that the isolates

that contained the genes iroN, ompTp, hlyF, and iss could have shared the same virulence

plasmid.

The isolates used for antimicrobial susceptibility testing were also assessed for specific

genes relating to heavy metal and antimicrobial resistance. Our study found the greatest presence

of the pcoD gene, followed by groEL, then aadA and aac3-VI. The pcoD gene is related to

copper resistance, groEl encodes a chaperone protein, aadA is associated with streptomycin

resistance, and aac3-VI is associated with gentamycin resistance (de Oliveria et al., 2020). A

previous study isolated E. coli from the clinical material at St. Bartholomew’s Hospital and

indicated a linear proportional relationship in the resistance patterns of select aminoglycoside

antimicrobials (Davies, 1971; Houang and Greenwood, 1977). This relationship indicates a cross

resistance between streptomycin, neomycin, tobramycin, and kanamycin; however, previous

studies found that E. coli isolates only showed a proportional increase in resistance between

tobramycin and gentamycin (Davies, 1971; Houang and Greenwood, 1977). Similarly, our study

found that streptomycin had one of the highest rates of resistance and there was a high

prevalence of the aadA gene. It is possible that the same prevalence of the genes aadA and aac3-

VI indicate that there could be cross-resistance to streptomycin and gentamycin.

CONCLUSION

In summary, the E. coli isolates taken from litter used by broiler chickens in an

experimental setting expressed phenotypic resistance to a wide range of antimicrobials from

different classes as well as genes contiguous to their virulence. Regarding antimicrobial

susceptibility, our isolates expressed moderate rates of resistance with majority of isolates
 Poultry E. coli Antimicrobial Resistance

resistant towards cepthalothin. Virulence genes such as iroN, ompT, and hlyF were most

prevalent in our isolates and frequently occurred simultaneously with one another, suggesting the

presence of a conserved virulence plasmidic region. The relevancy of zoonoses, especially now,

warrants greater efforts to investigate the intersection of agriculture and clinical medicine to not

only reduce economic losses for farmers and producers but to also advance the breadth and depth

of the One Health objective.

ACKNOWLEDGMENTS

Funding was provided by The Honors College Research Grant Program and the

Department of Animal and Avian Sciences at University of Maryland. The authors would like to

thank the members of the Weimer Lab from the University of Maryland and the Barbieri Lab

from the University of Georgia for their knowledge, guidance, and facilities that helped bring

this project to fruition.

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Table 1. Counts of E. coli isolates (n = 68) sourced from the litter of broiler chickens raised in

16 pens that were susceptible to all antibiotics and unique patterns of resistance to cephalothin

(KF), streptomycin (S), tetracycline (TET), ampicillin (AMP), colistin (CT), sulphonamides

(S3), and imipenem (IPM).

Antimicrobial # Isolates
AMP 1
KF 11
S 2
TET 2
AMP KF 2
KF S 4
CT KF 3
CT TET 1
TET KF 2
TET S 3
AMP KF S 1
AMP CT KF 1
CT TET KF 2
TET KF S 2
AMP S3 KF S 1
AMP TET KF S 2
AMP TET S3 KF 1
AMP CT S3 KF S 1
AMP CT TET KF S 1
AMP TET S3 KF S 2
AMP TET IPM S3 KF S 1
Total Susceptible 22
Total 68

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),
 Poultry E. coli Antimicrobial Resistance

CT (colistin), TET (tetracycline), 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.

Antimicrobial and Heavy Metal Genes as minimal

Antimicrobials
Resistance Genes predictors of APEC-like
O t t
g
ap s i p s a
aa
qac c i
o
h e a i
AP
A T I r c3 m i EC
ty C S K e e h( i n c u ISEc a Ed v r l t e r
Pen M E P S o - p s -
p P
T
T M
3 F t t
E
3) l tl o l 12 d
V
elt a o
T
y s
s
r e
like
B A IA P 1 D l A a1 C N F B J A
e L I p ?
Is
2 1 2 5 2 2 1 2 1 3 3 1 3 1
ol Prevale 1 8 2 7 5 8 1 7 3 1
0 3 7 4 9 0 8. 3 3 16. 7 13. 8 8 1 3 9 38.
at nce . . . . . . 7. . 8. .
. . . . . . 8 . . 2 . 2 . . . . . 2
e (%) 5 8 9 4 9 8 6 4 2 5
6 2 9 4 4 6 2 5 6 2 2 8 8 1
#
1 1 0 0 0 0 0 0 1 1 No
2 1 0 0 0 0 0 1 1 1 No
3 1 0 0 0 0 0 0 0 No
4 2 0 0 0 0 0 0 0 No
5 2 0 0 0 0 0 0 0 No
6 2 0 0 1 0 0 1 0 No
7 2 1 0 0 0 1 1 1 1 1 1 No
8 3 1 0 1 0 1 1 1 1 1 1 1 1 No
O
Ye
9 3 1 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1
5
s
O
10 3 7 0 0 1 0 0 1 1 No
5
11 3 0 0 0 0 0 0 0 1 1 1 No
O
Ye
12 4 9 1 0 0 0 0 1 1 1 1 1 1 1 1 1
1 s
O
Ye
13 4 9 1 0 1 0 1 1 1 1 1 1 1 1 1 1
1
s
O
Ye
14 4 7 1 0 1 0 1 1 0 1 1 1 1 1 1 1 1 1 1 1
8 s
O
Ye
15 4 7 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1
8
s
O
Ye
16 4 9 0 0 0 0 0 1 1 1 1 1 1 1 1 1
1
s
17 5 0 0 0 0 0 0 0 No
18 5 0 0 0 0 0 0 0 No
19 5 0 0 0 0 0 0 0 No
20 5 0 0 0 0 0 0 0 No
21 6 0 0 0 0 0 0 0 No
O
Ye
22 6 1 0 0 0 0 0 0 0 1 1 1 1 1 1 1
5
s
O
Ye
23 6 1 0 0 0 0 0 1 0 1 1 1 1 1 1
5
s
24 6 1 1 1 0 0 1 1 No
25 6 0 0 0 0 0 1 0 1 no
26 7 1 1 0 0 1 1 1 1 1 1 No
27 7 1 0 1 1 1 1 1 1 1 1 1 1 1 No
Ye
28 7 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1
s
29 8 0 0 0 0 0 0 0 1 No
30 8 0 0 0 0 0 1 0 No
Ye
31 8 1 0 0 0 0 1 0 1 1 1 1 1
s
 Poultry E. coli Antimicrobial Resistance

Ye
32 8 0 0 0 0 0 1 0 1 1 1 1 1
s
Ye
33 9 0 0 1 0 0 1 0 1 1 1 1
s
Ye
34 9 0 1 0 0 0 1 0 1 1 1 1 1 1 1 1
s
Ye
35 9 0 0 0 0 0 0 0 1 1 1 1 1 1 1
s
1
36 0 0 0 0 0 1 0 No
0
1
37 0 1 0 0 0 1 0 No
0
1
38 0 0 0 0 0 1 0 1 No
0
1
39 0 0 0 0 0 1 0 1 No
0
1
40
0
0 1 0 0 0 1 0 1 No
1
41 0 0 0 0 0 1 0 1 No
1
1 Ye
42 0 0 0 0 0 0 0 1 1 1 1 1
1 s
1
43 0 0 0 0 0 1 0 1 No
1
1
44 0 0 0 0 0 1 1 1 No
1
1
45 0 0 0 0 0 0 0 No
2
O
1 Ye
46 1 0 0 1 0 0 0 0 1 1 1 1 1 1 1 1 1
2 s
5
1 Ye
47 0 0 0 0 0 0 0 1 1 1 1 1 1 1
2 s
1
48 1 0 0 0 0 1 0 1 1 No
2
1
49 0 0 0 0 0 0 0 1 1 No
2
1
50 0 0 0 0 0 1 0 1 No
2
O
1 Ye
51 1 0 0 0 0 0 0 0 1 1 1 1 1
3 s
5
O
1 Ye
52 1 0 0 0 0 0 0 0 1 1 1 1 1 1
3 s
5
1
53
3
0 0 0 0 0 0 0 1 1 No
1
54 1 0 0 0 0 0 0 1 1 1 1 No
3
1 Ye
55 0 0 1 0 0 0 1 1 1 1 1 1 1 1
4 s
1 Ye
56 0 0 0 0 0 0 1 1 1 1 1 1 1 1
4 s
1 Ye
57
4
0 0 0 0 0 1 0 1 1 1 1 1
s
O
1 Ye
58 1 0 0 1 0 0 0 1 1 1 1 1 1 1 1
5 s
5
1
59 1 0 1 0 0 1 1 1 1 1 1 No
5
1 Ye
60 1 0 1 0 0 1 1 1 1 1 1 1
5 s
1 Ye
61 0 0 1 0 0 1 1 1 1 1 1 1
5 s
O
1 Ye
62 1 1 1 1 1 1 1
5 s
5
1
63 0 0 1 0 0 0 1 1 No
6
1
64 0 1 1 0 0 0 0 1 No
6
1
65
6
0 1 1 0 0 1 0 1 No
1
66 0 0 1 0 0 0 0 1 No
6
1
67 0 1 1 0 0 1 0 1 No
6
1
68 0 0 0 0 0 0 0 1 1 No
6
 Poultry E. coli Antimicrobial Resistance

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.

 Poultry E. coli Antimicrobial Resistance

25.0

20.0
% Prevalence

15.0

23.5
10.0 20.6
16.2 17.6 17.6
13.2 13.2
5.0 8.8 8.8
7.4 5.9
2.9
0.0
P

tl1

oD

ll
L
B

tA

IA

12

dA

1
I
-V

lta
su
oE

sil
tet

te

3)

Ec
in

aa
pc

c3

de
gr

h(

IS

aa

cE
ap

qa
Antibiotic and Heavy Metal Resistance Genes

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.
 Poultry E. coli Antimicrobial Resistance

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.