Molecular characterization of antibiotic resistant and potentially virulent enterococci isolated from swine farms and feed mills

Journal of Stored Products Research 77 (2018) 189e196

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Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr

Molecular characterization of antibiotic resistant and potentially virulent enterococci isolated from swine farms and feed mills Lakshmikantha H. Channaiah a, 1, Bhadriraju Subramanyam a, *, Ludek Zurek b a b

Department of Grain Science and Industry, Kansas State University, Manhattan, KS, 66506, USA Department of Entomology Kansas State University, Manhattan, KS, 66506, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 February 2018 Received in revised form 16 April 2018 Accepted 17 April 2018

A total of 108 swine feed samples were collected from six feed mills and two farms and tested for enterococcal contamination. Nearly 43% of these samples were positive for enterococci. The mean ± SE concentration of enterococci in feed samples ranged from 2.0
101 to 7.3
103 CFU/g of feed. About 38% of processed feed mill samples were contaminated with enterococci compared to 59% of swine farm samples. A total of 208 enterococcal isolates were represented by Enterococcus casseliflavus (54.8% of total isolates), E. gallinarum (17.8%), E. faecium (17.8%), E. hirae (5.8%), and E. faecalis (3.8%). These isolates were phenotypically resistant to tetracycline (48.5%), erythromycin (14.4%), streptomycin (13.4%), kanamycin (11.5%), ciprofloxacin (10.0%), ampicillin (2.8%), and chloramphenicol (1.4%). All isolates were susceptible to vancomycin and gentamicin. Tetracycline resistance was encoded by tetM gene (52.8%), tetO (14.4%), tetK (1.0%), and tetS (0.5%), whereas ermB conferred erythromycin resistance in 10.6% of all isolates. Several isolates carried genes coding for virulence factors, including gelatinase (gelE; 18.2%), an enterococcal surface protein (esp; 2.4%), and cytolysin (cylA; 2.4%). Only E. faecalis was b-hemolytic (2.9%) and gelatinolytic (3.4%). The aggregation substance gene asa1 was detected in 5 out of 8 E. faecalis isolates, of which four were phenotypically positive. The transposon Tn916/1545 was detected in 11.5% of all isolates. Mating assays revealed that 7 out of 8 E. faecalis could transfer tetM gene, and rate of transfer ranged from 2.0
103 to 1.6
105. The presence of antibiotic resistant and potentially virulent enterococci in swine farm samples and feed mill samples, though in low prevalence, raises concern and emphasizes the need for improved hygiene and quality standards on farms and in feed mills. © 2018 Elsevier Ltd. All rights reserved.

Keywords: Swine feed Enterococcus species Antibiotic resistance Virulence factors Gene transfer

1. Introduction Animal feed plays a significant role in the food supply chain. There are growing concerns regarding contamination of animal feed before arrival at and while on the livestock farm leading to transfer of foodborne pathogens such as Salmonella spp., Campylobacter spp., Yersinia spp., Listeria spp. and enterohaemorrhagic Escherichia coli, from food animals to humans (Dorn et al., 1975; Teuber, 1999; Bailar and Travers, 2002; Crump et al., 2002; Angulo et al., 2004; Hawkes and Ruel, 2006; Lester et al., 2006; Maciorowski et al., 2006; da Costa et al., 2007; EFSA, 2008). Animal feed can potentially become contaminated with

* Corresponding author. E-mail address: [email protected] (B. Subramanyam). 1 Current address: AIB International, 1213 Bakers Way, Manhattan, Kansas 66502, USA. https://doi.org/10.1016/j.jspr.2018.04.007 0022-474X/© 2018 Elsevier Ltd. All rights reserved.

food-borne pathogenic bacteria at several points throughout the feed production, including the primary production of feed ingredients, milling, mixing, extrusion, storage, and transportation (Cox et al., 1983; Hofacre et al., 2001; Kidd et al., 2002; Myint et al., 2007; Sapkota et al., 2007). Additionally, the use of a wide range of antimicrobial drugs representing all major classes of clinically important antimicrobials, from penicillin to third-generation cephalosporin compounds, in food animal production in the United States and many other countries (Silbergeld et al., 2008) has led to the emergence of antibiotic resistant strains of Enterococcus in food animals on farms (Aarestrup et al., 2000; Garcia-Migura et al., 2005; Ahmad et al., 2011; Novais et al., 2013). Therefore, the ingredients used in animal feed, and hygienic conditions of the processing environment and storage facilities are fundamentally important in terms of both the quality of the resulting food products and the potential animal or human health impacts associated with the animal-based food production systems (Crump et al., 2002; Maciorowski et al., 2006; da Costa et al., 2007; Sapkota

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et al., 2007). Enterococci traditionally have been considered to be of relatively low virulence in healthy individuals, but several species of enterococci have gained prominence in the last decade as the third leading cause of nosocomial infections in humans because of their resistance to several antibiotics, presence of virulence factors, and presence of an efficient horizontal gene transfer system (Coque et al., 1998; Gilmore, 2002; Gilmore et al., 2013). Enterococcal contamination of feed and feed ingredients by antibiotic-resistant Enterococcus species (de Costa et al., 2007; Ge et al., 2013), including vancomycin resistant E. faecium, has been reported (Schwalbe et al., 1999). There are increasing concerns of transfer of antibiotic resistant enterococcal strains from animal feed to humans through the food chain (Donabedian et al., 2003; Lester et al., 2006; Johnson et al., 2007; Hammerum et al., 2010). Additionally, the activity and by-products (fecal material) of storedproduct insects, birds, and rodents in the feed environment may increase the chance of pathogenic bacterial contamination (Daniels et al., 2003; Channaiah et al., 2010a). In our previous studies, we demonstrated that stored-product insects such as the red flour beetle, Tribolium castaneum (Herbst); confused flour beetle, Tribolium confusum Jacquelin du Val; warehouse beetle, Trogoderma variable Ballion; rusty grain beetle, Cryptolestes ferrugineus (Stephens); lesser grain borer, Rhyzopertha dominica (F.); drugstore beetle, Stegobium paniceum (L.); darkling beetle, Tenebrio molitor L; foreign grain beetle, Ahasverus advena (Waltl), and maize weevil, Sitophilus zeamais (Motschulsky), inhabiting United States feed mills carry antibiotic resistant and potentially virulent Enterococcus species in their gut (Larson et al., 2008; Channaiah et al., 2010a). Furthermore, we demonstrated that T. castaneum adults can act as potential vectors of antibioticresistant enterococci within the feed manufacturing environment, stressing the importance of proper pest management practices to reduce contamination of animal feed in feed mill environments (Channaiah et al., 2010b). However, it was unclear how insects acquire these enterococcal isolates. Therefore, to better understand the ecology of enterococci associated with animal feed, in the present investigation, feed samples collected from feed mills and swine farms were analyzed for enterococcal contamination to characterize their antibiotic resistance profiles. Specific research objectives of this investigation were to determine the prevalence, concentration, and diversity of antibiotic resistant and potentially virulent enterococci associated with animal feed. Additionally, the presence and transfer of mobile genetic elements in enterococci were examined. 2. Materials and methods 2.1. Collection of feed samples Feed samples were collected from six feed mills and two confined swine facilities located in four Midwestern United States (Kansas-KS, Indiana-IN, Iowa-IA, and Wisconsin-WI). A total of 108 feed samples (67 from feed mills and 41 from swine farms) were collected over a period of five months. Feed samples (250 g) from mills included raw materials as well as various processed fractions and finished feed. Samples of feed (250 g) from storage facilities were collected from swine farms. The complete list of feed samples collected in this study is shown in Table 1. All samples were collected in a sterile zipper-sealed plastic bag, labeled, and transported in a cooler to the laboratory for microbial analyses. 2.2. Isolation, enumeration, and identification of enterococci The feed sample in each sterile plastic bag was mixed manually

for 10 min to ensure thorough mixing of feed materials, and a representative sample (10 g) was suspended in 100 ml of phosphate buffered saline (PBS) (pH 7.2; MP Biomedicals, Santa Ana, California, USA), vortexed for 10 min and then dilution-plated on mEnterococcus agar (Difco Laboratories, Franklin Lakes, New Jersey, USA). Following incubation at 37  C for 48 h, the colony forming units (CFU) were counted to determine the enterococcal concentration per gram of feed. Morphologically different presumptive enterococcal colonies were cultured on trypticase soy agar (TSA) (Difco Laboratories) and confirmed at the genus level by presumptive esculin hydrolysis test using Enterococcosel broth (Difco Laboratories). Enterococcal species were identified by multiplex Polymerase Chain Reaction (PCR) using species-specific primer sets for E. faecalis, E. faecium, E. gallinarum, and E. casseliflavus (Kariyama et al., 2000). The strains used as positive and negative controls have been reported previously (Macovei and Zurek, 2006). Enterococcus hirae was identified by single PCR as described by Arias et al. (2006) and E. hirae ATCC 8043 was used as a positive control. The remaining unidentified isolates were identified by amplification and sequencing the manganese-dependent superoxide dismutase gene sodA (Poyart et al., 2000). 2.3. Phenotypic and genotypic screening of enterococci for antibiotic resistance For antibiotic susceptibility screening, antibiotics were selected based on their use in animal agriculture as well as in clinical treatments. Antibiotic susceptibility was determined for all identified enterococcal isolates by the disk-diffusion assay on MuellerHinton agar (Difco Laboratories) using seven different antibiotics on separate paper disks at the prescribed standard amounts: ampicillin (10 mg), ciprofloxacin (15 mg), tetracycline (30 mg), chloramphenicol (30 mg), erythromycin (15 mg), vancomycin (30 mg), and gentamicin (120 mg). Resistance to streptomycin (2000 mg/ml) and kanamycin (2000 mg/ml) were assessed by agar dilution technique on brain heart infusion (BHI; Difco Laboratories) agar. The disk-diffusion test is conducted by growing enterococcal lawn on the surface of a large (150 mm diameter) Mueller-Hinton agar plate. Seven different paper disks with fixed concentration, were placed on the inoculated agar surface and plates were incubated for 16e24 h at 35  C, after which the zones of growth inhibition around each of the antibiotic disks were measured to the nearest millimeter, and interpreted using the criteria published by the Clinical and Laboratory Standards Institute (CLSI, formerly the National Committee for Clinical Laboratory Standards or NCCLS) (CLSI, 2008, 2010). Routine quality control of antibiotic disks was performed using the E. faecalis ATCC 19433 strain. Single and multiplex PCR was performed to screen all identified isolates for tetracycline and erythromycin resistance genes. The group I multiplex reaction included the tetA, tetC, and tetQ genes, while the group II multiplex reaction included the tetM, tetS, tetK, and tetO genes (Ng et al., 2001; Villedieu et al., 2003). Single PCRs were used to screen tetW (Aminov et al., 2001) and ermB (Sutcliffe et al., 1996). The PCR reaction and conditions were described previously by Macovei and Zurek (2006). 2.4. Genotypic and phenotypic screening of enterococci for virulence determinants Enterococci possess several virulence factors such as enterococcal surface protein (esp), cytolysin activity (cylA), and gelatinase activity (gelE), encoded by transposons or mobile genetic elements. Enterococcal isolates were screened for the presence of virulence factors and transposons that can be ascribed to their roles in pathogenesis. Multiplex PCR was performed to screen all identified

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Table 1 Prevalence, concentration, and diversity of enterococci in feed samples collected from swine farms and feed mills. Source (State)

Diet

No. of samples Prevalence Mean conc.a

No. of isolates E. faecalis E. faecium E. gallinarum E. casseliflavus E. hirae

Lactating Prestarter Grower Gestation Finisher Feed mills (n ¼ 6) (KS, IN, IA, WI) Unprocessed L-lysine Calcium Trace minerals Selenium Salt Dried whey D Phosphatec Oat groats Soybean meal Soy þ corn Ground corn Ground oats Processed Prestarter Gestation Grower

41 8 9 6 9 9

24 (58.5) 4 5 4 6 5

5.4 ± 0.7
103 7.3 ± 0.3
103 7.2 ± 0.3
103 4.0 ± 0.4
103 3.7 ± 0.3
103 4.8 ± 0.4
103

160 12 53 42 30 23

6 1 3 1 0 1

29 2 8 5 8 6

29 6 6 11 3 3

85 3 32 18 19 13

11 0 4 7 0 0

51 2 3 6 1 1 2 3 2 15 11 1 4 16 8 4 4

16 (31.4) …b … … … … 1 … 1 6 5 1 2 6 (37.5) 3 1 2

1.8 ± 0.3
102 e e e e e 2.0 ± 0.0
101 e 2.0 ± 0.0
101 9.0 ± 2.5
101 1.8 ± 0.1
102 1.0 ± 0.0
102 3.0 ± 0.6
102 9.0 ± 0.3
101 2.0 ± 0.0
101 1.0 ± 0.0
102 1.5 ± 0.3
102

39 0 0 0 0 0 2 0 2 15 11 4 5 9 3 2 4

2 0 0 0 0 0 0 0 0 1 0 0 1

6 0 0 0 0 0 1 0 0 1 2 2 0 2 0 1 1

26 0 0 0 0 0 1 0 2 13 6 1 3 3 1 1 1

1 0 0 0 0 0 0 0 0 0 1 0 0

0 0 0

4 0 0 0 0 0 0 0 0 0 2 1 1 4 2 0 2

Total (%)

108

46 (42.6)

208

8 (3.8)

37 (17.8)

37 (17.8)

114 (54.8)

12 (5.8)

Swine farms (n ¼ 2) (KS)

a b c

b

b

0 0 0

CFU±SE/g feed sample. Not detected. Dicalcium phosphate.

isolates for four putative virulence determinants: asa1 (aggregation substance), esp (enterococcal surface protein), gelE (gelatinase) and cylA (cytolysin) (Vankerckhoven et al., 2004), that play critical role in pathogenesis such as colonization of the host, biofilm formation, and destruction of red blood cells during infection. These isolates were also tested for gelatinase activity on Todd Hewitt agar (Difco Laboratories) with 2% skim milk, expression of the asa1 gene (only in E. faecalis) using clumping assay, and cytolysin expression by b-hemolysis on Columbia blood agar base (Difco Laboratories, Detroit, MI) supplemented with 5% human blood (Rockland Immunochemicals, Limerick, Pennsylvania, USA) using suitable positive control strains as described previously (Macovei and Zurek, 2006).

2.5. Screening enterococci for mobile genetic elements and conjugation assays Mobile genetic elements such as transposons play a pivotal role in the dissemination of antibiotic resistance in enterococci. Therefore, all identified isolates were screened by PCR for integrase gene (int) for detection of Tn916/1545 conjugative family transposons (Gevers et al., 2003) that frequently carry the tetM gene. Enterococcus faecalis OG1RF:pCF10 was used as a positive control. Broth and filter mating experiments were carried out as described by Ike et al. (1998) and Tendolkar et al. (2006), respectively, to determine the mobility of transposon Tn916/1545 containing tetM gene. The donor strains were E. faecalis isolated in this study (tetracycline, MIC ¼ Minimum inhibitory concentration was 40 mg/ml) and the recipient strain was E. faecalis OG1SSp (streptomycin, MIC ¼ 2000 mg/ml). Both assays were performed with a donor and a recipient ratio of 1:10. After allowing mating for 4 h in broth and 16 h on filter paper, the mixed culture was dilution plated on BHI agar supplemented with suitable combinations of antibiotics and incubated for 24e48 h at 37  C. The transfer frequency for each isolate was calculated as the number of transconjugants per

donor and recipient CFU. The presence of the tetM gene in transconjugants was confirmed by PCR. 3. Results 3.1. Enterococcal diversity in animal feed The 108 feed samples (41 from swine farms and 67 from feed mills) yielded 208 enterococcal isolates, of which 160 isolates were recovered from the two swine farms located in Kansas and the remaining 48 were isolated from feed samples collected from six feed mills located in Kansas, Indiana, Iowa, and Wisconsin. About 59 and 38% of processed feed samples from two swine farms and six feed mills, respectively, were positive for enterococci. The mean ± SE concentration of enterococci in feed samples ranged from 2.0 ± 0.0
101 to 7.3 ± 0.7
103 CFU/g of feed (Table 1). Enterococcus casseliflavus (54.8% of total isolates) was the dominant enterococcal species followed by E. gallinarum (17.8%), E. faecium (17.8%), E. hirae (5.8%), and E. faecalis (3.8%). Among the samples collected from feed mills, a majority of the enterococci (81.3%, 39 out of 48 isolates) were isolated from unprocessed feed (raw ingredients) compared to processed and finished feed (18.75%, 9 out of 48). 3.2. Antibiotic resistance profile and prevalence of the tet and erm genes About 58% of all isolates were resistant to at least one antibiotic. Isolates were phenotypically resistant to tetracycline (48.5%), followed by erythromycin (14.4%), ciprofloxacin (10.0%), streptomycin (13.4%), kanamycin (11.5%), ampicillin (2.8%), and chloramphenicol (1.4%) (Table 2). None of the isolates was resistant to vancomycin or gentamicin. Few enterococcal isolates showed resistance to two or more antibiotics: E. casseliflavus (13.1%), E. gallinarum (43.0%), E. faecium (24.3%), E. hirae (66.6%), and E. faecalis (50.0%). The

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Table 2 Antibiotic resistance profiles of enterococci isolated from feed samples collected from feed mills and swine farms. Source

Number of isolates showing resistance for antibioticsa

Number of isolates

Feed mills E. faecalis E. faecium E. gallinarum E. casseliflavus E. hirae

48 2 8 8 29 1

Swine farms E. faecalis E. faecium E. gallinarum E. casseliflavus E. hirae

160 6 29 29 85 11

Total (%)

208

Amp

Tet

Chl

Cip

Kan

—b e e 2 1

2 5 7 4 1

1 e e e 1

1 e 1 e 1

e e 3 1 e

e

2 1 e

6 21 25 22 8

e e e 1 e

1 e 6 10 1

6 (2.8)

101 (48.5)

3 (1.4)

21 (10.0)

——

Str

Ery

e

4 4 e

e 1 e 2 e

1 e 12 4 3

e 3 10 4 3

e 9 5 9 4

24 (11.5)

28 (13.4)

30 (14.4)

—

No resistance was detected to vancomycin and gentamicin. a Amp ¼ Ampicillin; Tet ¼ Tetracycline; Chl ¼ Chloramphenicol; Cip ¼ ciprofloxacin; Kan ¼ kanamycin; Str ¼ streptomycin; Ery ¼ erythromycin. b Not detected.

antibiotic resistant combinations of Tetr-Strr (10.5%) were most common, followed by the combination of Tetr-Cipr (8.6%), and TetrEryr (7.7%). Enterococcal isolates in this study harbored the tetracycline resistance genes tetM (52.8%, 110 out of 208 isolates), tetO (14.4%), tetK (1.0%), and tetS (0.5%) (Table 3). The tetA, tetC, tetQ, and tetW genes were not detected. Ten isolates carried multiple tet genes (tetM and tetO). The ribosomal protection mechanism encoded by tetM was detected most frequently in ascending order in E. faecalis, E. faecium, E. casseliflavus, and E. gallinarum. The tetO gene was found mainly in E. casseliflavus. The ermB gene was detected in 10.6% of total isolates and was more common in E. casseliflavus and E. faecium.

E. faecalis harbored the asa1 gene. Out of all isolates, 24% possessed at least one virulence factor, and a few isolates (4.8%) possessed two or more virulence factors. Only E. faecalis (87.5%, 7 out of 8) showed strong gelatinase activity as well as b-hemolytic activity (75%, 6 out of 8). The clumping assay showed that 4 out of 5 E. faecalis were positive for aggregation substance. All other isolates belonging to E. gallinarum, E. casseliflavus, E. faecium, and E. hirae were negative for the phenotypic assays. 3.4. Prevalence of Tn916/1545 conjugative transposons and conjugal transfer The Tn916/1545 conjugative transposon family was detected in 24 (11.5%) of all isolates (Table 3) mostly in E. faecalis and E. faecium. Out of the 24 isolates, most of these transposons (91.6%) carried either tetM or ermB. Conjugal assays showed that only one isolate (1 out of the 8 E. faecalis isolates) could transfer tetM in broth mating. However, filter mating assay followed by PCR confirmation

3.3. Virulence profile The gelE gene was detected most frequently (18.2%) followed by cylA (2.4%), asa1 (2.4%), and esp (2.4%) (Table 3). Five out of eight

Table 3 Prevalence of virulence traits, tetracycline and erythromycin resistance genes, and Tn916/1545 family of transposons of enterococci from feed samples from swine farms and feed mills. Source

Number of isolates Virulence genes

Virulence phenotype

TET genesb

ERY genesc Transposon

gelE

asa1

esp

cylA

Gelatinase Hemolytic Aggra

tetM

tetS

tetK

tetO

ermB

Tn916/1545

Feed mills E. faecalis E. faecium E. gallinarum E. casseliflavus E. hirae

2 8 8 29 1

2 2 2 4 e

2 e e e e

—d e 1 e e

2 e e e e

2 e e e e

2 e e e e

1 NA NA NA NA

2 7 8 e e

e e e e e

e 1 e e e

e e e 1 e

1 3 e 6 e

2 3 2 1 e

Swine farms E. faecalis E. faecium E. gallinarum E. casseliflavus E. hirae

6 29 29 85 11

5 4 12 7 e

3 e e e e

e e 1 3 e

3 e e e e

5 e e e e

4 e e e e

3 NA NA NA NA

6 26 28 31 2

e e e 1 e

e e e 1 e

e 5 e 22 2

1 4 2 3 2

6 5 3 2 e

Total (%)

208

38 (18.2) 5 (2.4) 5 (2.4) 5 (2.4) 7 (3.4)

6 (2.9)

4 (1.9) 110 (52.8) 1 (0.48) 2 (0.96) 30 (14.4) 22 (10.6)

NA ¼ Not applicable. a Aggr. ¼ Aggregation substance. b TET ¼ Tetracycline resistance genes. c ERY ¼ Erythromycin resistance genes. d Not detected.

24 (11.5)

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revealed that 6 out of 8 E. faecalis isolates transferred tetM to the recipient strain with transfer rate ranging from 2.0
103 to 1.6
105 transconjugants per donor (or 2.6
104 to 1.0
109 transconjugants per recipient). 4. Discussion An earlier study investigated S. enterica contamination in swine feed in the United States (Molla et al., 2010). To the best of our knowledge the information on enterococcal contamination of swine feed and feed ingredients in the United States is limited in the literature. In the present study, we characterized enterococcal isolates from 108 samples collected from feed mills and swine farms across the Midwest region of the United States. The enterococcal contamination levels in feed samples collected from swine farms were higher compared to that from the feed mills. Detection of enterococci in about 37.5% (6 out of 16) of the processed samples from commercial feed mills suggests cross-contamination after extrusion or pelleting at high temperatures during processing possibly due to unhygienic handling, packaging, distribution, and storage. Additionally, the pelleting process, where temperatures exceed 82  C, may not kill all bacterial cells, especially enterococci, as they are tolerant to various stresses including mild heat treatment (Ahmad et al., 2002). As expected the processed feed (finished feed) were less contaminated than the unprocessed ones. de Costa et al. (2007) analyzed poultry feed and detected viable Enterococcus species in all tested feed samples and 66% of raw feeding materials with a median value of 2.7 log CFU/g. Recently, the United States Food and Drug Administration surveillance studies revealed that 87% of animal feed ingredients, comprising animal and plant byproducts, were contaminated with various Enterococcus species (Ge et al., 2013). In our samples, we found the majority of isolates as E. casseliflavus followed by E. gallinarum, E. faecium, E. hirae, and E. faecalis. The use of plant by-products such as forage, grains, plant protein products, and by-products of fruits in feed manufacturing process may be the likely source of E. casseliflavus, E. gallinarum, and E. hirae in the feed samples (Muller et al., 2001; Fisher and Phillips, 2009). Enterococcus casseliflavus has been isolated as the major enterococcal species from fruits and vegetables (McGowan et al., 2006). In general, the use of animal by-products such as meat meal, blood meal, feather meal, bone meal, fish meal, egg shell meal, bone marrow, and dried poultry litter, as feed ingredients are the likely sources of enterococci in feed ingredients, especially E. faecalis and E. faecium (Kinley et al., 2010; Ge et al., 2013). However, Ge et al. (2013) recovered E. faecium, based on only phenotypic tests, as the dominant species in the plant by-products. Furthermore, activities of stored-product insects, birds and rodents are common in feed mills (Pellitteri et al., 1983; Larson et al., 2008) and may contribute to the fecal contamination of the feed with clinically important species of Enterococcus such as E. faecalis and E. faecium. Recycling of rendered animals and animal waste as well as addition of antibiotics and organochemicals to the animal feed may make the feed itself a source of antibiotic resistant bacterial strains (Davis and Roberts, 1999; da Costa et al., 2007). The high frequency of resistance to tetracycline and erythromycin in enterococci reported in the present study could be due to the widespread use of these antibiotics in animal feed for growth promotion and therapeutic purposes (Witte, 1998; van den Bogaard and Stobberingh, 1999; Sapkota et al., 2007). Resistance to kanamycin and streptomycin can be attributed to the use of aminoglycosides in animal feeds to manage intestinal infections (Riviere and Spoo, 2001). Although the use of enrofloxacin has been banned in poultry feed in the United States in 2005, it is approved for therapeutic use in

193

swine. Prevalence of ciprofloxacin-resistant enterococci in our swine feed samples is similar to that reported previously in Campylobacter coli isolates from swine (Delsol et al., 2004). The low prevalence of chloramphenicol resistance in enterococcal isolates was expected because chloramphenicol is no longer allowed in animal feed in the United States (FARAD, 2014). Although the use of penicillin and ampicillin is approved for use in swine and poultry feeds (FDA-CVM, 2007), risk assessment studies show current usage presents very low impact on human health (Cox et al., 2009). The low prevalence of resistance reported in the present study (ampicillin, 2.8%) and in others such as penicillin 1.1% (Ge et al., 2013) do not raise much concern. None of our isolates was resistant to vancomycin and gentamicin. Avoparcin, an analogue of vancomycin, has never been used in animal feed in the United States (Coque et al., 1996; FARAD, 2014). Only a few studies reported vancomycin-resistant enterococci in livestock environments in the United States, for example, in chicken feed (Schwalbe et al., 1999) and in swine feces (Donabedian et al., 2010). The resistance frequencies reported in our study for tetracycline, erythromycin, chloramphenicol, vancomycin, and gentamicin were comparable to the findings of Ge et al. (2013), except for low prevalence of ciprofloxacin (7.2%), streptomycin (1.9%), and kanamycin (0%). Genotypic analysis showed that tetM and tetO were most common in tetracycline-resistant isolates, whereas ermB was common in erythromycin resistant ones. These tet genes are widespread among antibiotic-resistant enterococci of food animal and food origin (Fairchild et al., 2005; Macovei and Zurek, 2006; Ahmad et al., 2011). Stine et al. (2007) sampled swine and their environment, including feed from a confined animal feeding facility and reported high prevalence of tetM (85%) and tetS (50%); however, the contribution of individual sample sources was not discussed. Macrolide resistance mediated by erm genes is common in Streptococcus, Staphylococcus, Enterococcus, and other food-borne pathogens (Pyorala et al., 2014), and prevalence of ermB genes in agricultural environment has been reported in European countries as well as in the United States (Jensen et al., 1999; Loch et al., 2005; Edrington et al., 2014). This is because erythromycin is widely used in animal feed as a therapeutic agent to treat infections caused by Gram-positive bacteria (Erskine, 2000). The clinical strains of Enterococcus are well known for their association with endocarditis, bacteremia, and urinary tract infections due, in part, to the presence gelE and esp (Gilmore et al., 2002). We found a higher percentage of gelE in enterococci compared to gelatinase producers, and this may be due to the presence of silent genes that are expressed only under in vivo conditions as suggested in earlier studies (Creti et al., 2004; Biavasco et al., 2007; Macovei et al., 2009; Ahmad et al., 2011). The highly pathogenic esp-positive E. faecium isolates are a worldwide human-health threat (Willems et al., 2005). We detected the esp gene in only a few E. gallinarum and E. casseliflavus isolates. However, esp positive E. faecalis, E. faecium, and E. casseliflavus have been reported from food and food-animal environments (Willems et al., 2005; Biavasco et al., 2007; Macovei and Zurek, 2007; Ahmad et al., 2011). Hemolysin is another virulence factor found in as many as 60% of E. faecalis clinical strains (Gilmore et al., 2002). We reported that all E. faecalis that harbored the cytolysin factor (cylA) were hemolytic, which is in agreement to a previous finding where the E. faecalis, E. faecium, and E. casseliflavus of food-animal and insect origin showed a strong correlation between the presence of cylA and b-hemolysis on human blood (Ahmad et al., 2011). b-hemolytic strains of E. faecalis also have been isolated from meat and poultry (Poeta et al., 2006; Barbosa et al., 2009). We showed E. faecalis strains with cylA also had asa1 as they reside on pheromoneresponsive transferable plasmids, such as pAD1 (Gilmore et al., 2002; Creti et al., 2004). These potentially virulent E. faecalis

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strains were further selected for the conjugal transfer experiments. In this study, antibiotic resistance and virulence in enterococci was more frequently detected in the feed samples collected from farms compared to that from the feed mills. This suggested that the farm environment likely plays a major role in the contamination. The presence of antibiotic resistance and virulence genes in E. casseliflavus, E. gallinarum, and E. hirae cannot be ignored. Although uncommon in clinical settings, they have been associated with sporadic cases of meningitis, prosthesis-associated infection, and endocarditis (Iaria et al., 2005; Cooper et al., 2008; Talarmin et al., 2011), and have the potential to transfer traits to other nosocomial pathogens such as E. faecalis and E. faecium if suitable conditions prevail. Conjugative transposons Tn916/1545 encoding ermB and tetM have been reported in food animals (swine and broiler) as well as in human patients (De Leener et al., 2004; Kresken et al., 2004; Cauwerts et al., 2007). These transposons are efficient in transferring resistance genes to other bacteria due to their high integration ability into the host chromosome or plasmids (Clewell et al., 1995). Conjugation experiments showed that the filter mating assay was more efficient in intra-species transfer of the tetM gene from E. faecalis strains when compared to broth mating assay. Mobilization of transposon such as Tn916, if present on the chromosome, needs close contact of the donor and recipient bacterial cells on a solid surface, such as a filter paper (Ike et al., 1998; Clewell and Dunny, 2002). Transconjugants were confirmed by amplification of the tetM gene and suggested its location on Tn916/1545. However, additional experiments need to be performed to find the precise location of the mobile genetic element on a chromosome or plasmid. Interestingly, enterococcal species diversity reported in our previous studies on stored-product insects from feed mills (Larson et al., 2008; Channaiah et al., 2010a) is similar to that of feed analyzed in this study. Moreover, the antibiogram and virulence profile of Enterococcus species in these studies were highly comparable. This further points to the potential of insects to acquire the antibiotic resistant bacteria from the feed and cross-contaminate the feed through their feeding and defecating activities (Channaiah et al., 2010b; Zurek and Ghosh, 2014). The hygienic condition, post-lethality handling practices, and ecology of feed storage warehouses, feeding operations, and pest management on farms may have a direct impact on the presence and diversity of antibiotic-resistant bacteria (Singer et al., 2007; Wellington et al., 2013). Therefore, our data reinforces the need for improved quality standards in feed mills and animal farms to prevent further contamination of feed before it is consumed by farm animals and thus prevent antibiotic-resistant enterococci from entering the animal food and human food supply chain. Acknowledgements The authors wish to thank all the cooperating swine farms and feed mill companies for allowing us to collect feed samples. This paper is contribution number 18e314-J of the Kansas State University Agricultural Experiment Station. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jspr.2018.04.007. References Aarestrup, F.M., Agerso, Y., Gerner-Smidt, P., Madsen, M., Jensen, L.B., 2000. Comparison of antimicrobial resistance phenotypes and resistance genes in

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