Molecular characterization of antimicrobial-resistant Enterococcus faecalis and Enterococcus faecium isolated from layer parent stock

Molecular characterization of antimicrobial-resistant Enterococcus faecalis and Enterococcus faecium isolated from layer parent stock

∗

,∗ Kwang Won Seo,∗ Jong Bo Shim,† Se hyun Son,∗ Eun Bi Noh,∗ and Young Ju Lee∗,1

College of Veterinary Medicine and Zoonoses Research Institute, Kyungpook National University, Daegu 41566, Republic of Korea; and † Korean Poultry TS Co., Ltd., Incheon 17415, Republic of Korea erythromycin-resistant E. faecalis isolates, which was higher than that of 32 (71.7%) erythromycin-resistant E. faecium isolates. Twenty-one high-level gentamicinresistant Enterococcus spp. (17 E. faecalis and 4 E. faecium) carried at least one aminoglycoside-modifying enzyme gene, aac(6 )Ie-aph(2 )-Ia or ant(6)-Ia. Fourteen isolates that harbored both aac(6 )Ie-aph(2 )-Ia and ant(6)-Ia exhibited pattern A with IS256 at both ends. Ten high-level ciprofloxacin-resistant Enterococcus spp. (8 E. faecalis and 2 E. faecium) showed amino acid changes from serine to isoleucine at codons 83 in gyrA, and 80 in parC. Also, the virulence genes ace, asa1, efaA, and gelE were detected in this study. To the best of our knowledge, this is the first study to examine the prevalence and characteristics of antimicrobial-resistant E. faecalis and E. faecium isolates in the layer parent stock. Our findings support the need for a surveillance program to monitor the emergence of antimicrobialresistant E. faecalis and E. faecium in layer operating system.

ABSTRACT Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) are ubiquitous intestinal bacteria in humans and animals that can easily acquire antimicrobial resistance, which allows them to have roles as antimicrobial resistance indicators. In addition, layer parent stock produces thousands of eggs for the production of commercial laying hens and can transfer a variety of viral and bacterial agents to chicks. The objective of this study was to determine the prevalence and characteristics of antimicrobial-resistant E. faecalis and E. faecium isolated in the layer parent stock level of the egg-layer operating system in South Korea. A total of 129 E. faecalis and 166 E. faecium isolates from 74 flocks of 30 layer parent stock were tested for resistance in this study. The prevalence of doxycycline- (51.9%), erythromycin- (53.5%), high-level gentamicin- (13.2%), high-level kanamycin- (31.0%), high-level streptomycin(30.2%), and tetracycline- (64.3%) resistant E. faecalis isolates were higher than those for E. faecium isolates (P < 0.05). The ermB gene was detected in 66 (95.7%)

Key words: Enterococcus faecalis, Enterococcus faecium, layer parent stock, antimicrobial resistance 2019 Poultry Science 0:1–8 http://dx.doi.org/10.3382/ps/pez288

INTRODUCTION

in cattle, diarrhea in swine and cattle, and septicemic diseases in poultry (Frye et al., 2006; Jackson et al., 2011; Larsson et al., 2014; Nowakiewicz et al., 2016; Wu et al., 2016). E. faecalis and E. faecium are able to produce potential virulence factors that may enhance their pathogenicity, in other words responsible for causing diseases (Kim et al., 2016; Yılmaz et al., 2016). Moreover, E. faecalis and E. faecium have a highly developed ability to acquire resistance genes from the same or different species via transferable plasmids or transposons (Chajecka-Wierzchowska et al., 2017).  In the poultry industry, the layer operating system has a pyramidal structure with the grandparent stock at the top followed by parent-stock flocks that produce eggs for the production of commercial laying hens. The presence of antimicrobial-resistant microorganisms in fecal material of the parent stock can be subsequently disseminated into the environment via the intestine and transferred to chicks through hatcheries serving as

The use of antimicrobials combined with strict biosecurity and hygiene measures has helped the poultry industry to control the spread of infectious pathogens and increase feed efficiencies (Engberg et al., 2000). However, antimicrobial-resistant bacteria are becoming increasingly common and are causing a global health problem (Lee et al., 2003). Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) are members of the gastrointestinal biota of humans and animals, and are considered opportunistic pathogens that are mainly responsible for nosocomial infections in humans and many types of infections in animals, such as mastitis  C 2019 Poultry Science Association Inc. Received February 13, 2019. Accepted May 1, 2019. 1 Corresponding author: [email protected]

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Yeong Bin Kim

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MATERIALS AND METHODS

Hinton agar, using the following antibiotic disks (BD Biosciences): ampicillin (10 μg), chloramphenicol (C, 30 μg), ciprofloxacin (CIP, 5 μg), doxycycline (30 μg), erythromycin (E, 15 μg), penicillin (10 units), rifampin (RA, 5 μg), tetracycline (TE, 30 μg), and vancomycin (30 μg). Susceptibility results were interpreted in accordance with Clinical and Laboratory Standards Institute guidelines (CLSI) guidelines (CLSI, 2013). Multidrug resistance (MDR) was defined as acquired non-susceptibility to 3 or more antimicrobial categories. The minimum inhibitory concentration (MIC) was obtained by performing standard agar dilution methods with Mueller-Hinton agar (BD Biosciences) in accordance with the recommendations of the CLSI (CLSI, 2013). The MIC breakpoint values for high-level CIP (≥64 μg/mL), high-level gentamicin (≥500 μg/mL), and high-level streptomycin (≥2000 μg/mL) were defined by the CLSI, whereas the high-level kanamycin (≥500 μg/mL) was defined by the Soci´et´e Fran¸caise de Microbiologie (CASFM, 2011).

Sampling From 2016 to 2018, dust and fecal were periodically collected from 74 flocks of a 30-layer parent stock in South Korea. A total of 2 varieties of feces and 3 varieties of dust were sampled at 20 wk of age for each flock in accordance with the standards set by the National Poultry Improvement Plan (USDA, 2012). The dust samples were swabbed from dusty areas by using a gauze (12 layers, 10 cm × 10 cm), which moistened with sterile skim milk. The fecal samples (approximately 10 g fecal droppings) were collected randomly at the selected sampling site. The samples were individually inoculated into buffered peptone water (BD Biosciences, Sparks, MD, USA) and incubated for 18 to 24 h at 37◦ C.

Bacterial Isolation Isolates were identified as enterococci by using established methods (Simjee et al., 2002). Pre-enriched buffered peptone water was transferred to the Enterococcosel broth (BD Biosciences) at a 1:10 ratio and incubated for 18 to 24 h at 37◦ C. After enrichment, the bacteria-containing Enterococcosel broth was streaked onto an Enterococcosel agar (BD Biosciences). At least 3 typical colonies on the Enterococcosel agar were selected and subsequently identified as E. faecalis or E. faecium by using the polymerase chain reaction (PCR) method as previously described (Dutka-Malen et al., 1995; Ta et al., 1999). If some isolates from the same origin showed the same antimicrobial susceptibility patterns, only one isolate was randomly chosen and included in this study.

Antimicrobial Susceptibility Testing The antimicrobial susceptibility test was performed by applying the disk diffusion method in Muller-

Molecular Analysis PCR amplification was performed using primers for the C-resistance genes (cfr, fexA, and optrA), Eresistance genes (ermA, ermB, and mef), TE-resistance genes (tetL, tetM, and tetO), and aminoglycoside modifying enzyme (AME) genes [aac(6 )-Ie–aph(2 )Ia, aph(2 )-Ib, aph(2 )-Ic, aph(2 )-Id, ant(3 )-Ia, and ant(6)-Ia] (Clark et al., 1999; Vakulenko et al., 2003; Kehrenberg and Schwarz, 2006; Di Cesare et al., 2013; Choi and Woo, 2015). The following virulence genes, ace, asa1, cylA, efaA, esp, gelE, and hyl, were also identified by using PCR as described previously (Choi and Woo, 2013).

Detection of IS256-flanking Pattern for aac(6 )Ie-aph(2 )-Ia The IS256-flanking patterns were detected for highlevel gentamicin-resistance (HLGR) Enterococcus spp. harboring aac(6 )Ie-aph(2 )-Ia. Polymerase chain reaction using 2 primer pairs as reported by Watanabe et al (2009) was performed to determine the IS256-flanking patterns.

Screening for Mutations in gyrA and parC The PCR amplification of the quinolone resistancedetermining regions of gyrA and parC was carried out using a method previously described (Kwak et al., 2013). The amplified DNA was purified using a PCR purification kit (Qiagen, Valencia, CA) and then sequenced (Bioneer, Daejeon, Korea). The DNA sequences were compared with those of the standard gyrA (GenBank Accession No. AF060881) and parC (GenBank Accession No. AB017811) genes (El Amin et al., 1999).

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reservoirs (Kroˇcko et al., 2012; Osman et al., 2018). In South Korea, antimicrobial drugs such as aminoglycoside, ß-lactam, and fluoroquinolone have been widely used (Kang et al., 2010). However, aminoglycoside, the 3rd and 4th cephalosporins, and fluoroquinolones are antibiotics classified by the World Health Organization as “critically important in human medicine” due to their importance for treating pathogenic infections (WHO, 2015). Although the characteristics of antimicrobial-resistant bacteria from commercial layer farms or eggs have been described in South Korea (Lee et al., 2010; Im et al., 2015), those in layer parent stock has not been fully described. Therefore, this study was to determine the characteristics of antimicrobialresistant E. faecalis and E. faecium isolated from layer parent stock level of the layer operating system in South Korea.

ENTEROCOCCI FROM LAYER PARENT STOCK

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Statistical Analysis Statistical analysis was performed using the Statistical Package for Social Science version 23 (SPSS; IBM, Korea). The unpaired t-test was used to identify statistically significant differences in the numbers of antimicrobial resistance phenotypes and genotypes between E. faecalis and E. faecium. Differences were considered significant at P < 0.05.

RESULTS Distribution of Antimicrobial Resistance of E. faecalis and E. faecium A total of 295 isolates of enterococci were recovered from the feces and dust samples from 30 farms. A comparison of the antimicrobial resistances in the 129 E. faecalis and 166 E. faecium isolates from the parent stock of the layer operating system is presented in Figure 1. The prevalence of doxycycline-, E-, and TEresistant E. faecalis isolates among the total E. faecalis isolates were 51.9%, 53.5%, and 64.3%, respectively, and those proportions were significantly higher (P < 0.05) than those for the E. faecium isolates. Moreover, the prevalence of HLGR (13.2%), high-level kanamycinresistance (31.0%), high-level streptomycin-resistance (30.2%), and MDR- (38.0%) E. faecalis isolates were also higher than those of the E. faecium isolates (P < 0.05). On the other hand, the prevalence of antimicrobial resistance to RA (62.1%) in E. faecium isolates was significantly higher (P < 0.05) than that in E. faecalis.

Table 1 summarizes the prevalence of antimicrobial resistance genes in E. faecalis and E. faecium isolates. A total of 7 (70.0%) and 2 (20.0%) of the 10 C-resistant E. faecalis isolates were positive for the optrA and fexA genes, respectively. However, one (20.0%) of the 5 Cresistant E. faecium isolates were positive for the optrA gene only. The ermB gene was detected in 66 (95.7%) of the 69 E-resistant E. faecalis, which was significantly higher (P < 0.05) than that of 32 (71.7%) among 45 E-resistant E. faecium. The tetM gene was the most common tet resistance gene detected and was present in 86.0% of the TE-resistant Enterococcus spp. In addition, tetM was detected together in isolates that had the tetL gene. The tetL gene was detected in 59 (71.1%) of the 83 TE-resistant E. faecalis isolates, which was significantly higher (P < 0.05) than that among the 46 TE-resistant E. faecium (17/46, 37.0%). Also, 51 E. faecalis isolates (51/57, 89.5%) simultaneously possessed the ermB, tetL, and tetM genes.

Characteristics of HLGR E. faecalis and E. faecium The characteristics of the 21 HLGR Enterococcus spp. isolates (17 E. faecalis and 4 E. faecium) are summarized in Table 2. All HLGR isolates carried at least one AME gene, aac(6 )Ie-aph(2 )-Ia or ant(6)Ia. A total of 14 of the HLGR isolates harbored both aac(6 )Ie-aph(2 )-Ia and ant(6)-Ia and showed pattern A with insertion sequence IS256 at both ends. All HLGR isolates had kanamycin MIC >2,048 ug/mL and

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Figure 1. Prevalence of antimicrobial resistance of 129 E. faecalis and 166 E. faecium from layer parent stock. AM, ampicillin; C, chloramphenicol; CIP, ciprofloxacin; DOX, doxycycline; E, erythromycin; P, penicillin; RA, rifampin; TE, tetracycline; VA, vancomycin; HLGR, high-level gentamicin; HLK; high-level kanamycin; HLS, high-level streptomycin; MDR, multidrug-resistance. ∗ There were significantly differences (P < 0.05) between E. faecalis and E. faecium.

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Table 1. Prevalence of the antimicrobial resistance genes in chloramphenicol, erythromycin or tetracycline-resistant E. faecalis and E. faecium from layer parent stock. Number of isolates carrying the gene/Number of resistant isolates (%) Chloramphenicol

E. faecalis E. faecium Total 1

Tetracycline

Erythromycin+ Tetracycline

cfr

fexA

optrA

ermB1

mef

tetL1

tetM

tetO

tetM+tetL1

tetM+tetO

ermB+tetM

ermB+tetL+tetM1

0/10 (0.0) 0/5 (0.0) 0/15 (0.0)

2/10 (20.0) 0/5 (0.0) 2/15 (13.3)

7/10 (70.0) 1/5 (20.0) 8/15 (53.3)

66/69 (95.7) 32/45 (71.1) 98/114 (86.0)

0/65 (0.0) 0/45 (0.0) 0/114 (0.0)

59/83 (71.1) 17/46 (37.0) 76/129 (58.9)

75/83 (90.4) 36/46 (86.0) 111/129 (86.0)

9/83 (10.8) 6/46 (13.0) 15/129 (11.6)

59/83 (71.1) 17/46 (37.0) 76/129 (58.9)

5/83 (6.0) 6/46 (13.0) 11/129 (8.5)

2/57 (3.5) 0/22 (0.0) 2/79 (2.5)

51/57 (89.5) 6/22 (27.3) 57/79 (72.2)

There were significant differences (P < 0.05) between E. faecalis and E. faecium.

gentamicin MIC ranged from 1,024 to >2,048 ug/mL. For streptomycin, 19 isolates showed MIC ≥ 2,048 ug/mL. HLGR Enterococcus spp. with E- and TEresistance were the most common, and they harbored one or 2 of the TE-resistance genes (tetL, tetM, and tetO) or the E-resistance gene (ermB). A total of 4 virulence genes (ace, asa1, efaA, and gelE) were detected among the HLGR isolates carrying one or more of the virulence genes. However, the cyl, esp, and hyl genes were not detected in any of the HLGR isolates.

Characteristics of High-level CIP-resistant E. faecalis and E. faecium The characteristics of 10 high-level CIP-resistant (HLCR) Enterococcus spp. isolates (8 E. faecalis and 2 E. faecium) are summarized in Table 3. All HLCR isolates showed amino acid changes from serine to isoleucine at codons 83 in gyrA and 80 in parC. All HLGR isolates had CIP MIC range from 64 to >128 ug/mL and ENR MIC 64 ug/mL. All HLCR isolates also showed resistance to E and MDR and all harbored ermB. However, 7 of the 9 C-resistant isolates harbored fexA and/or optrA genes, and 5 of the 6 TE-resistant isolates harbored both tetL and tetM genes. A total of 7 of the HLCR isolates possessed at least one of the following three virulence genes, ace, asa1, or efaA.

DISCUSSION Antimicrobial resistance in bacteria isolated from commercial laying hens and eggs has been reported worldwide, but the study of antimicrobial resistance at the parent-stock level of the layer operating systems is a relatively new field. This study showed the prevalence and characteristics of antimicrobial resistance in E. faecalis and E. faecium from the parent-stock level of the layer operating systems in South Korea. In this study, of the 295 isolates, 129 E. faecalis (43.7%) and 166 E. faecium (56.3%) were recovered. In the previous study, these species were predominance in the poultry environment (Hayes et al., 2004).

The resistance to doxycycline, E, and TE in E. faecalis was significantly higher (P < 0.05) than those in E. faecium. Macrolides and tetracyclines have been widely used in the poultry industry because of their broad activity spectra (S´ anchez Valenzuela et al., 2013; APQA, 2017). The mechanism of TE-resistance has been described as an effect on efflux pumps (tetL) and ribosomal protection proteins (tetM and tetO) (Choi and Woo, 2015), whereas E-resistance has been associated with the presence of ermB. These genes can be easily transferred by conjugative transposons such as the Tn916/1545-like family (Agersø et al., 2006; Tremblay et al., 2011). A previous study reported that the Tn916/1545-like family was observed more frequently in investigated strains of E. faecalis than those of E. faecium (Mikalsen et al., 2015). Furthermore, the Tn916/1545-like family has been associated with pheromone-responsive plasmids in E. faecalis, which can accelerate their transfer among E. faecalis strains (Huys et al., 2004). For this reason, the prevalence of MDR is higher in E. faecalis than E. faecium. In addition, the optrA gene which encodes an ATP-binding cassette transporter and confer transferable resistance to phenicols, and the cfr gene which confers resistance to phenicols (Wang et al., 2015) were detected in this study. The spread of these genes could significantly limit treatment options for MDR bacteria (Liu et al., 2012; Tamang et al., 2017). In this study, the prevalence of RA-resistant E. faecium was significantly higher (P < 0.05) than that for E. faecalis. This result is similar to that in a previous study that investigated the poultry industry in South Korea (Kim et al., 2018a). A previous study described an isolate of E. faecium that developed RA-resistance without any evidence of mutations in the rpoB gene, enzymatic inactivation, or efflux pump (Miller et al., 2015). This result indicated that RA-resistant E. faecium have already spread and persist in poultry environments, regardless of the presence or absence of the rpoB gene. In this study, the prevalence of HLAR was higher in E. faecalis isolates than in E. faecium isolates. This result is similar to that previously reported for an investigation into the poultry industry in South Korea

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Species

Erythromycin

Farm1/Flock2

Farm7/Flock3

Farm12/Flock1

Farm17/Flock1

Farm18/Flock1

Farm18/Flock2

Farm18/Flock3

Farm18/Flock3

Farm20/Flock3

Farm20/Flock3

Farm24/Flock2

Farm25/Flock2

Farm26/Flock1

Farm26/Flock2

Farm26/Flock2

Farm13/Flock2

Farm21/Flock2

Farm23/Flock3

Farm27/Flock3

F1–2-T-2

F5–24-G-21

F8–1-G-21

F9–2-G-21

F10–2-G-2

F10–4-G-1

F10–4-T-2

F10–6-G-1

F11–1-G-1

F11–1-G-2

F12–18-G-1

F12–30-G-1

F13–2-G-1

F13–4-T-2

F13–6-G-1

E. faecium F8–12-G-11

F11–4-G-1

F12–2-T-1

F14–1-T-2

A A A A A A A A A

aac(6’)Ie-aph(2 )-la, ant(6)-Ia aac(6’)Ie-aph(2 )-la, ant(6)-Ia aac(6’)Ie-aph(2 )-la, ant(6)-Ia aac(6’)Ie-aph(2 )-la, ant(6)-Ia aac(6’)Ie-aph(2 )-la, ant(6)-Ia aac(6’)Ie-aph(2 )-la, ant(6)-Ia aac(6’)Ie-aph(2 )-la, ant(6)-Ia aac(6’)Ie-aph(2 )-la, ant(6)-Ia aac(6’)Ie-aph(2 )-la, ant(6)-Ia

NT A A A

ant(6)-Ia aac(6’)Ie-aph(2 )-la, ant(6)-Ia aac(6’)Ie-aph(2 )-la, ant(6)-Ia aac(6’)Ie-aph(2 )-la, ant(6)-Ia

aac(6’)Ie-aph(2 )-la, ant(6)-Ia

A

A

aac(6’)Ie-aph(2 )-la, ant(6)-Ia



D

aac(6’)Ie-aph(2 )-la

D

aac(6’)Ie-aph(2 )-la NT2

D

aac(6’)Ie-aph(2 )-la

ant(6)-Ia

D D

IS256-flankig pattern

aac(6’)Ie-aph(2 )-la aac(6’)Ie-aph(2 )-la

AME gene

RA

DOX-E-TE

DOX-E-TE

C-CIP-E

C-DOX-ETE CIP-E-RATE

DOX-E-TE

DOX-E-TE

DOX-E-TE

DOX-ERA-TE DOX-E-TE

DOX-E-TE

DOX-E-TE

DOX-E-TE

DOX-E-TE

C-CIPDOX-E-TE

C-CIP-E

RA-TE DOX-ERA-TE DOX-RATE C-CIP-E

2

ermB, optrA ermB, tetL, tetM ermB, tetL, tetM NT

ermB, optrA ermB, optrA ermB, fexA, optrA, tetL, tetM ermB, tetL, tetM ermB, tetL, tetM ermB, tetL, tetM ermB, tetL, tetM ermB, tetL, tetM ermB, tetL, tetM ermB, tetL, tetM ermB, tetL, tetM ermB, tetL, tetM ermB, tetL, tetM ermB, tetL, tetM

tetO, tetM ermB, tetL, tetM tetM

Antimicrobial resistance genotype

ace, asa1, efaA ace, asa1, efaA, gelE ace, asa1, efaA, gelE ace, asa1, efaA, gelE

ace, asa1, efaA, gelE asa1, gelE

asa1, gelE

ace, asa1, efaA, gelE ace, asa1, efaA, gelE ace, asa1, efaA, gelE ace, asa1, efaA, gelE ace, asa1, efaA, gelE ace, asa1, efaA, gelE ace, asa1, efaA, gelE asa1, gelE

ace, asa1, efaA ace, asa1, efaA ace, efaA

gelE

gelE gelE

Virulence genes

> 2048

> 2048

> 2048

2048

> 2048

> 2048

> 2048

> 2048

> 2048

> 2048

> 2048

> 2048

> 2048

> 2048

> 2048

2048

2048

1024

> 2048

2048 > 2048

G

S > 2048 > 2048

2048 > 2048 > 2048 < 256

> 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 < 256 > 2048 > 2048 > 2048

K > 2048 > 2048 > 2048 > 2048 > 2048 > 2048

> 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048 > 2048

MIC (ug/ml)4

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These isolates are included in Table 3 as well. NT, not target. 3 C, chloramphenicol; CIP, ciprofloxacin; DOX, doxycycline; E, erythromycin; TE, tetracycline; RA, rifampin. 4 G, gentamicin; K, kanamycin; S, streptomycin.

1

Farm1/Flock1 Farm1/Flock2

Farm/flock

E. faecalis F1–1-T-1 F1–1-T-2

Species/strains

Antimicrobial resistance phenotype3

Table 2. Characteristics of 21 high-level gentamicin-resistant E. faecalis and E. faecium isolated from layer parent stock.

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6 These isolates are included in Table 2 as well. AM, ampicillin; C, chloramphenicol; DOX, doxycycline; E, erythromycin; HLG, high-level gentamicin; HLK, high-level kanamycin; HLS, high-level streptomycin; TE, tetracycline; RA, rifampin. 3 ND, not detected. 4 CIP, ciprofloxacin; ENR, enofloxacin. 2

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16 64 64 > 128 S83I S83I Farm10/Flock3 Farm13/Flock2

S80I S80I

E-RA C-E-HLG-HLK

ermB ant(6)-Ia, ermB, optrA

ND ace, asa1, efaA

64 64 64 64 64 64 64 64 64 64 64 64 > 128 > 128 > 128 > 128 S83I S83I S83I S83I S83I S83I S83I S83I

E. faecalis F4–1-T-1 F4–2-T-1 F4–6-G-1 F5–24-G-21 F8–1-G-21 F9–1-G-2 F9–1-T-2 F9–2-G-21 E. faecium F7–12-G-1 F8–12-G-11

Farm6/Flock1 Farm6/Flock2 Farm7/Flock1 Farm7/Flock3 Farm12/Flock1 Farm15/Flock2 Farm15/Flock2 Farm15/Flock2

S80I S80I S80I S80I S80I S80I S80I S80I

C-DOX-E-TE C-DOX-E-TE C-DOX-E-RA-TE C-E-HLG-HLK-HLS C-E-HLG-HLK-HLS C-DOX-E-TE C-DOX-E-TE C- DOX-E-HLG-HLK-TE

ermB, optrA. tetL, tetM, ermB, tetL, tetM ermB aac(6 )-Ie–aph(2 )-Ia, ermB, optrA ant(6)-Ia, ermB, optrA ermB, fexA, optrA, tetL, tetM ermB, optrA, tetL, tetM aac(6 )-Ie–aph(2 )-Ia, ermB, fexA, optrA, tetL, tetM

ND3 ND ace, efaA ace, asa1, efaA ace, asa1, efaA ace, efaA ace, efaA ace, efaA

ENR CIP Virulence genes Antimicrobial resistance genotype Species/strains

Farm/flock

gyrA

parC

Antimicrobial resistance phenotype2 Amino acid change

Table 3. Characteristics of 10 high-level ciprofloxacin-resistant E. faecalis and E. faecium isolated from layer parent stock.

(Han et al., 2011). Aminoglycoside is frequently used in the poultry industry, and gentamicin is commonly injected subcutaneously with either Marek’s or infectious bursal disease vaccines into day-old chicks in hatcheries (APQA, 2017; Kim et al., 2018b). Enterococcus spp. are intrinsically resistant to aminoglycosides and the acquired high-level resistance to aminoglycoside can be associated with plasmid-mediated AME genes. The presence of AME genes such as aac(6 )-Ie–aph(2 )-Ia and ant(6)-Ia implies that gentamicin cannot be synergistically used with glycopeptide or ß-lactam for the treatment of enterococcal infection (Chow, 2000; Udo et al., 2004). In addition, previous studies have identified aac(6 )-Ie–aph(2 )-Ia as part of a transposon, designated as truncated Tn5281, that lacks IS256 at either the 5 - or 3 -end (Feizabadi et al., 2008; Rosvoll et al., 2012). For the Tn5281-like elements, the IS256-flanking patterns A with IS256 at both ends and pattern D without IS256 at both ends detected in the present study were previously detected in clinical isolates of enterococci (Simjee et al., 1999; Klibi et al., 2006). The presence of an insertion sequence in the Enterococcus spp. implies that these elements are being spread by horizontal gene transfer (Mikalsen et al., 2015). In this study, 17 E. faecalis and 4 E. faecium isolates were identified as HLGR isolates that harbored aac(6 )-Ie–aph(2 )-Ia and ant(6)-Ia genes. Moreover, 11 of the 16 E. faecalis isolates carrying the aac(6 )Ie–aph(2 )-Ia gene were observed to exhibit pattern A with IS256 at both ends. This result suggests that HLGR E. faecalis may be transferred among species, and HLGR E. faecalis are predominant than HLGR E. faecium in poultry industry in South Korea. Fluoroquinolone is also commonly used in the poultry industry, and enrofloxacin is a broad-spectrum fluoroquinolone antibiotic currently approved for the treatment of domestic animals in South Korea. In this study, 8 E. faecalis and 2 E. faecium isolates were identified as HLCR isolates. High-level acquired resistance results from point mutations in gyrA and parC genes encoding the A subunits of DNA gyrase and topoisomerase IV, respectively (Cattoir and Giard, 2014). In this study, all HLCR E. faecalis and E. faecium isolates showed mutations in both gyrA and parC, and the ranges of CIP and enrofloxacin MIC values were 64 to 128 ug/mL and 16 to 64 ug/mL, respectively. Also, except for one isolate, all HLCR E. faecalis and E. faecium isolates showed MDR. Previous studies have suggested that the rampant use of fluoroquinolones has contributed to the emergence of very high-level or complete resistance and a high prevalence of MDR (Ahmed et al., 2006; Kim et al., 2018a). Such observations have also been reported in the previous studies of human cases of enterococcal urinary tract infections (Kim and Woo, 2017; Leavis et al., 2006), thus, supporting a previous suggestion that the use of enrofloxacin in poultry may lead to the development of fluoroquinolone-resistant bacteria that can infect humans (Kim et al., 2018a, b).

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MIC (ug/ml)4

KIM ET AL.

ENTEROCOCCI FROM LAYER PARENT STOCK

ACKNOWLEDGMENTS This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Agriculture, Food and Rural Affairs Research Center Support Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (716002-7).

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In this study, the HLCR and HLGR Enterococcus spp. isolates showed enrichment of virulence-associated gene prevalence. Most HLCR and HLGR Enterococcus spp. isolates possessed common virulence genes: ace (collagen-binding protein), asa1 (aggregation substance), and efaA (cell wall-associated protein involved in immune evasion), but only the HLGR isolates possessed gelE (gelatinase). Previous studies reported that the majority of virulence genes were associated with the presence of clinically important resistance genes coding for antimicrobials (Aslam et al., 2012; Chajecka-Wierzchowska et al., 2017; McGowan-Spicer  et al., 2008). The presence of virulence genes does not necessarily mean that the strains isolated from foods of animal origin cause diseases in humans, but it may indicate a pathogenic potential as these factors have been found to contribute to infection severity (Yılmaz et al., 2016). This is the first study to investigate the prevalence and characteristics of antimicrobial-resistant Enterococcus spp. isolated from layer parent stock. The results further indicate the need for a surveillance program to monitor the emergence of antimicrobial-resistant Enterococcus spp. in layer operating systems.

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