Antimicrobial Resistance,Virulence,and Phylogenetic Characteristics of Escherichia coli Isolates from Clinically Healthy Swine

Abstract

A total of 344 commensal Escherichia coli isolates from clinically healthy pigs were examined for antimicrobial resistance phenotypes, class 1 integrons, resistance genes, virulence gene profile, and phylogenetic groups. The majority of E. coli isolates were resistant to tetracycline (96.2%) and ampicillin (91.6%). Up to 98% were multidrug resistant. Seventy-three percent of the isolates carried class 1 integrons. Inserted-gene cassette arrays in variable regions included incomplete sat, aadA22, aadA1, dfrA12-aadA2, and sat-psp-aadA2, of which the aadA2 gene cassette was most prevalent (42.9%). Horizontal transfer was detected in eight E. coli isolates carrying class 1 integrons with dfrA12-aadA2 gene cassette array. Sixteen resistance genes were identified among the E. coli isolates with corresponding resistance phenotype. Ten virulence genes (including elt, estA, estB, astA, faeG, fasA, fedA, eaeA, paa, and sepA) were detected, of which fasA was most commonly found (98.3%). Most of the E. coli isolates belonged to phylogenetic group B1. Significantly positive associations were observed between some virulence genes and some resistance phenotypes and genotypes (p<0.05). The results support a finding that commensal E. coli have a role as reservoirs for antimicrobial resistance–encoding genes and virulence determinants.

Introduction

E scherichia coli is one of the predominant-commensal inhabitants of small intestine of pigs. Most E. coli strains are harmless, but some serotypes are implicated in various human and animal diseases. Pigs are food animals in most parts of the world and serve as one of the major reservoirs for this bacterium. Therefore, E. coli in healthy pigs may be a potential health risk if it is transmitted to humans and contributes to cross-contamination among carcasses during slaughter and post-slaughter processing (Diarrassouba et al., 2007).

As antimicrobial therapy is an important tool for treatment of bacterial infections, E. coli infections are becoming increasingly difficult to treat due to emergence of the multiresistant strains (Yang et al., 2009; Wang et al., 2011). Pathogenic E. coli require multiple virulence factors to cause diarrhea; however, nonpathogenic strains from clinically healthy pigs were reported to carry a number of virulence genes (Schierack et al., 2006). The latter are considered normal microflora since they do not cause apparent diseases. Both resistance and virulence genes have been identified on transmissible genetic elements, allowing their horizontal transfer among E. coli and other bacterial species (Dobrindt et al., 2002; Moreira et al., 2005).

Like in other bacteria, antimicrobial resistance is more common among pathogenic than commensal E. coli (Boerlin et al., 2005). This could be linked to the more intense and frequent exposure of pathogenic strains to antimicrobials and/or the association of resistance and virulence determinants (Boerlin et al., 2005). Based on phylogenetic background, it was shown that antimicrobial resistance among the E. coli animal isolates was not related to decreased virulence features (Johnson et al., 2003). Conversely, the good correlation between resistance to certain antibiotics and decreased virulence traits was reported in the human isolates (Johnson et al., 2003). Despite several investigations, there is no definite answer for clarification of all these linkages. To date, there are only a few published studies on the association among antimicrobial resistance, virulence factors, and phylogroups in E. coli isolates from healthy pigs (Schierack et al., 2006; Rosengren et al., 2009).

Emergence of multiresistant E. coli strains carrying virulence factors is a particular concern due to their possible dissemination into the environment through manure spreading, and consequently to farm workers and food processing plants. The aims of this study were to investigate the prevalence and association of antimicrobial resistance, virulence factors, and phylogenetic groups of E. coli from clinically healthy pigs.

Materials and Methods

Bacterial isolates

Three hundred forty-four E. coli isolates were obtained from fecal samples directly collected per rectum from clinically healthy pigs confirmed by farm veterinarians, during 2007–2008, in six farms in central Thailand (i.e., Ratchaburi [n=92] and Chonburi [n=110]) and in northeast Thailand (i.e., Buriram [n=43], Nakhonratchasrima [n=39], and Udon Thani [n=69]). The pigs were 4–18 weeks old. All the E. coli strains were isolated at Veterinary Diagnostic Laboratory (VDL), Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand primarily for a screening and diagnosis project of Novartis (Thailand) Ltd. Animal Health Business Unit, Bangkok and kept in the VDL-strain collection. The E. coli strains were isolated on MacConkey (Quinn et al., 1994) and identified on Eosin Methylence Blue agar and/or classical biochemical methods (Carter and Cole, 1990). One colony from each positive sample was collected and stored as 20% glycerol stocks at −80°C. The complete records on antimicrobial use were not available in all farms. Based on the information provided by farm veterinarians, the antimicrobials routinely used included amoxicillin, chlortetracycline, tylosin, tiamulin, and fosfomycin.

Antimicrobial susceptibility testing

The standard twofold agar dilution technique was used to determine minimum inhibitory concentrations (MICs) of all the E. coli isolates to nine antimicrobial agents (CLSI, 2008). An E. coli isolate resistant to three or more separate classes of antimicrobials was defined as multidrug resistance (Sahm et al., 2001). E. coli American Type Culture Collection (ATCC) 25922 was used as quality control organism.

Detection of class 1 integrons and resistance determinants

All E.coli isolates were examined for the presence of class 1 integrons using polymerase chain reaction (PCR) (Chuanchuen et al., 2007; Ekkapobyotin et al., 2008). Inserted-gene cassettes were characterized in all the intl1-positive isolates using conserved segment (CS) PCR with 5′ CS and 3′ CS primers (Levesque et al., 1995). The nucleotide sequences were analyzed by comparison with those available in the GenBank Database. CS-PCR amplicons were grouped based on their sizes and digested with at least two different restriction endonuclease enzymes, including AluI, EcoRI, MseI, and BamHI. The PCR amplicons with the same restriction patterns were considered identical.

Eighteen resistance genes were screened by PCR in all resistant isolates according to their resistance phenotypes (Chuanchuen et al., 2007; Chuanchuen and Padungtod, 2009).

Determination of mutations in gyrA and parC

Fourteen ciprofloxacin-resistant E. coli isolates were randomly selected and investigated for mutations in the quinolone resistance-determining region (QRDR) in gyrA and parC (Chuanchuen and Padungtod, 2009). The nucleotide sequences were compared with those deposited in GenBank (accession no. X06373 for gyrA and accession no. M58408 for parC).

Determination of virulence genes

All the E. coli isolates were screened for the presence of 14 virulence genes using multiplex PCR with specific primer pairs (Bosworth and Casey, 1997; Ngeleka et al., 2003; Boerlin et al., 2005; Chapman et al., 2006). The virulence genes tested included elt, estA, estB, astA, stx2e, faeG, fanA, fasA, fedA, fimF4, eaeA, paa, aidA, and sepA.

Conjugation experiments

All the E. coli isolates carrying class 1 integrons with resistance gene cassettes (n=46) were assayed for integron transfer (Chen et al., 2004). Conjugation recipients were the spontaneous rifampicin-resistant derivatives of Salmonella Enteritidis strain SE12 (MIC=256 μg/mL). SE12 is susceptible to all antimicrobials tested and does not harbor class 1 integrons and plasmid. Transconjugants were confirmed to be Salmonella by growth on xylose lysine deoxycholate (XLD) agar (Sparks, Difco, MD) containing rifampicin (32 μg/mL) and an appropriate antibiotic, i.e., trimethoprim (10 μg/mL) or streptomycin (50 μg/mL), and screened for class 1 integrons corresponding to those in the E. coli donors.

Phylogenetic analysis

Triplex PCR specific for three genetic markers chuA, yjaA, and TSPE4.C2 were performed to determine phylogenetic groups of all the E. coli isolates (Clermont et al., 2000) and phylo-groups were assigned as previously described (Gordon et al., 2008).

Statistical analysis

Comparisons of the associations between antimicrobial-resistance phenotypes, resistance genes, virulence genes, and phylogenetic groups were performed separately by using Pearson's Chi-square test (SPSS, version 17.0). A p-value of<0.05 was considered statistically significant. Odds ratios (Ors) and 95% confidence intervals (CIs) were calculated.

Results

Antimicrobial resistance phenotypes

Ninety-nine percent of E. coli were resistant to at least one antimicrobial agent (Table 1). The highest resistance percentages were to tetracycline (96.2%) and ampicillin (91.6%). Frequencies of resistance to streptomycin, chloramphenicol, trimethoprim, sulfamethoxazole, gentamycin, and ciprofloxacin were 82.6%, 79.4%, 79.4%, 67.4%, 63.4%, and 52.3%, respectively. Ninety-eight percent were multidrug-resistant, whereas 16% were resistant to all antimicrobials tested. Forty-one resistance patterns were observed, of which AMP-CHP-CIP-GEN-STR-SUL-TET-TRI (15.7%) was most common.

Table 1.

Antimicrobial Resistance, Resistance-Encoding Genes, and Phylogenetic Groups of Escherichia coli Isolates (n=344)

	Phylogenetic group of resistant isolates, no. (%)				Phylogenetic group of resistance genes					Resistance gene
Resistance phenotype^a (no. of isolates.)	B1 (n=282)	B2 (n=24)	D (n=10)	U^b (n=28)	Gene	B1 (n=282), no. (%)	B2 (n=24), no. (%)	D (n=10), no. (%)	U^b (n=28), no. (%)	Gene	No. (%) of isolates
Ampicillin (315)	257 (91.1)	23 (95.8)	10 (100)	25 (89.3)	bla_PSE1	0 (0)	0 (0)	0 (0)	0 (0)	bla_PSE1 only	0 (0)
					bla_TEM	239 (84.8)	23 (95.8)	10 (100)	25 (89.3)	bla_TEM only	297 (94.3)
Chloramphenicol (273)	212 (57.2)	23 (95.8)	10 (100)	28 (100)	cat_A	182 (64.5)	23 (95.8)	10 (100)	28 (100)	cat_A only	10 (3.7)
					cat_B	10 (3.5)	0 (0)	0 (0)	0 (0)	cat_B only	9 (3.3)
					cmlA	61 (21.6)	14 (58.3)	0 (0)	10 (35.7)	cmlA only	167 (61.2)
										cat_B, cmlA	76 (27.8)
										At least one	262 (96)
Ciprofloxacin (180)	151 (53.6)	5 (20.8)	10 (100)	14 (50)	nd	nd	nd	nd	nd	nd	nd
Gentamicin (218)	184 (65.3)	16 (66.7)	10 (100)	8 (25.6)	aadB	73 (25.9)	11 (45.8)	0 (0)	2 (7.2)	aadB only	13 (6)
					aadA1	167 (59.2)	16 (66.7)	10 (100)	8 (28.6)	aadA1 only	14 (6.4)
					aadA2	147 (52.1)	16 (66.7)	10 (100)	8 (28.6)	aadA2 only	4 (1.8)
										aadB, aadA1	10 (4.6)
										aadA1, aadA2	114 (52.3)
										aadB, aadA1, aadA2	63 (28.9)
										At least one	218 (100)
Streptomycin (284)	222 (78.7)	24 (100)	10 (100)	28 (100)	aadA1	195 (69.1)	23 (95.8)	10 (100)	27 (96.4)	aadA1 only	7 (2.5)
					aadA2	162 (57.4)	24 (100)	10 (100)	26 (92.9)	aadA2 only	3 (1.1)
					strA-strB	194 (68.8)	10 (41.7)	10 (100)	19 (67.9)	strA-strB	21 (7.4)
										aadA1, aadA2	41 (14.4)
										strA-strB, aadA1	34 (12.0)
										strA-strB, aadA2	5 (1.8)
										strA, strB, aadA1, aadA2	173 (60.9)
										At least one	284 (100)
Sulfamethoxazole (232)	189 (67)	19 (79.2)	0 (0)	24 (85.7)	sul1	2 (0.7)	9 (37.5)	0 (0)	0 (0)	sul2 only	10 (4.3)
					sul2	64 (22.7)	9 (37.5)	0 (0)	10 (35.7)	sul3 only	129 (55.6)
					sul3	159 (56.4)	10 (41.7)	0 (0)	22 (78.6)	sul1, sul2	11 (4.7)
										sul2, sul3	62 (26.7)
										At least one	212 (91.4)
Tetracycline (331)	269 (95.4)	24 (100)	10 (100)	28 (100)	tetA	210 (74.5)	24 (100)	10 (100)	17 (60.7)	tetA only	184 (55.6)
					tetB	110 (39)	3 (12.5)	0 (0)	10 (35.7)	tetB only	46 (13.9)
										tetA, tetB	77 (23.3)
										At least one	307 (92.8)
Trimethoprim (273)	211 (74.3)	24 (100)	10 (100)	28 (100)	dfrA1	22 (7.8)	7 (29.2)	0 (0)	10 (35.7)	dfrA1 only	12 (4.4)
					dfrA10	68 (24.1)	10 (41.7)	0 (0)	9 (32.1)	dfrA10 only	32 (11.7)
					dfrA12	163 (57.8)	15 (62.5)	0 (0)	13 (46.4)	dfrA12 only	119 (43.6)
										dfrA1, dfrA12	17 (6.2)
										dfrA10, dfrA12	45 (16.5)
										dfrA1, dfrA10, dfrA12	10 (3.7)
										At least one	235 (86.1)

Break points (μg/mL) and range of concentrations (μg/mL) of antimicrobials were ampicillin (32, 1–512), chloramphenicol (32, 1–512), ciprofloxacin (4, 0.125–256), gentamicin (8, 0.25–256), streptomycin (32, 1–256), sulfamethoxazole (512, 1–2048), tetracycline (16, 1–512), and trimethoprim (16, 1–512).

U strains that were not assigned to a phylogenetic group.

nd, not determined.

Class 1 integrons and their transfer

Two hundred fifty-one (72.9%) E. coli isolates were positive to intI1, of which 56 isolates (22.3%) carried class 1 integrons containing variable regions with size ranging from 650 to 2,700 base pair (bp; Table 2). Inserted-gene cassette arrays included incomplete sat, aadA22, aadA1, dfrA12-aadA2, and sat-psp-aadA2. The predominant resistance gene cassette was aadA2 (42.9%). The aadA1 gene cassette array was most commonly found (26.8%).

Table 2.

Class 1 Integrons in Escherichia coli Isolates (n=56)

IP ^a	Size (bp)	Gene cassettes	No. (%) of isolates	Antimicrobial resistance pattern (no. of isolates)	Virulence gene profile (no. of isolates)
I	650	Incomplete sat	10 (17.9)	AMP-CHP-CIP-STR-SUL-TET-TRI (10)	astA, elt, estB, fasA (10)
II	1000	aadA1	15 (26.8)	CHP-STR-TET-TRI (3)	elt, fasA (4), astA, elt, estB, faeG, fasA, sepA (10)
				AMP-GEN-STR-TET-TRI (3)	eaeA, elt, estA, fasA, paa (1)
				AMP-GEN-SUL-TET-TRI (1)
				CHP-GEN-STR-TET-TRI (1)
				AMP-GEN-STR-SUL-TET-TRI (4)
				AMP-CHP-GEN-STR-SUL-TET-TRI (1)
				AMP-CIP-GEN-STR-SUL-TET-TRI (1)
				AMP-CHP-CIP-GEN-STR-SUL-TET-TRI (1)
III	1000	aadA22	7 (12.5)	AMP-CHP-GEN-STR-TRI (1)	elt, fasA (7)
				AMP-CHP-GEN-STR-TET-TRI (3)
				AMP-CHP-GEN-STR-SUL-TET-TRI (3)
IV	2000	dfrA12-aadA2	14 (25.0)	AMP-CIP-GEN-STR-TET-TRI (1)	elt, fasA (5), astA, elt, fasA (1), astA, fasA (6)
				AMP-CHP-CIP-GEN-STR-TET-TRI (4)	astA, faeG, fasA (2)
				AMP-CHP-CIP-GEN-STR-SUL-TET-TRI (9)
V	2700	sat-psp-aadA2	10 (17.9)	AMP-CHP-GEN-STR-SUL-TET-TRI (10)	elt, fasA (10)

Integron profile.

AMP, ampicillin; CHP, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; STR, streptomycin, SUL, sulfamethoxazole, TET, tetracycline; TRI, trimethoprim.

Class 1 integrons with dfrA12-aadA2 array in eight E. coli isolates were conjugally transferred to Salmonella recipients. All the Salmonella transconjugants were confirmed to harbor the corresponding class 1 integrons. and acquired trimethroprim and streptomycin resistance phenotypes of the E. coli donors (data not shown).

Distribution of antimicrobial resistance genes and mutations in gyrA and parC

Seventeen resistance genes, except bla _PSE-1, were identified (Table 1). Most resistant strains were positive to at least one resistance gene tested. Some carried multiple genes encoding for an identical resistance phenotype.

Eleven out of 14 ciprofloxacin-resistant isolates carried at least a single point mutation in gyrA (10 isolates) and/or parC (11 isolates). Three point mutations (including C-248-T, G-259-A, and A-281-C) were detected in gyrA, resulting in amino acid substitutions Ser-83-Leu, Asp-87-Asn, and Gln-94-Pro in GyrA, respectively. Seven isolates (MIC 8-128 μg/mL) carried double mutations (i.e., Ser-83-Leu and Asp-87-Asn) in GyrA. Two isolates (MIC 16 and 64 μg/mL) carried only one point mutation (i.e., Ser-83-Leu or Asp-87-Asn) in GyrA, and one isolate (MIC 32 μg/mL) contained all three amino acid substitutions in GyrA. Nine isolates with a gyrA mutation (except one strain carrying double mutations, MIC 8 μg/mL) additionally harbored a G-173-T mutation in parC, leading to Ser-58-Ile in ParC. Two isolates with a ParC mutation (MIC 32 and 128 μg/mL) lacked GyrA mutation.

Virulence gene profiles

Ten out of 14 virulence genes were detected in all the E. coli isolates, including fasA (98.3%), elt (57.9%), astA (34.9%), estB (25%), paa (22.7%), fedA (16.9%), sepA (14.5%), eaeA (13.1%), faeG (4.7%), and estA (0.6%) (Table 3). All carried at least one virulence gene. None were positive to stx2e, fanA, aidA, and fimF41. Thirty virulence gene profiles were defined, of which elt, fasA was most prevalent (28.2%) (Table 4).

Table 3.

Association Among Virulence Genes Among Escherichia coli in Phylogenetic Group B1 (n=282)

		Association between virulence genes ^a
Virulence genes(n)	No. (%) isolates in phylogroup B1 (n=282)	elt	estB	astA	faeG	fedA	eaeA	paa	sepA
elt	146 (51.8)	ns	—	2.2 (1.4–3.6)	7.1 (1.6–31.9)	—	—	0.6 (0.3–1.0)	2.0 (1.1–3.9)
estA	2 (0.7)	—	—	—	—	—	—	—	—
estB	76 (27)	—	ns	39.7 (17.0–92.8)	9.5 (3.0–30.4)	221 (50.6–965.3)	—	19.5 (10.1–37.7)	2.6 (1.4–4.9)
astA	110 (39)	2.2 (1.4–3.6)	39.7 (17.0–92.8)	ns	27.0 (3.5–207.6)	26.8 (10.2–70.5)	—	4.5 (2.6–7.8)	3.2 (1.7–5.9)
faeG	16 (5.7)	7.1 (1.6–31.9)	9.5 (3.0–30.4)	27.0 (3.5–207.6)	ns	na	—	—	44.7 (9.8–205.0)
fasA	281 (99.5)	—	—	—	—	—	—	—	—
fedA	54 (19.2)	—	221 (50.6–965.3)	26.8 (10.2–70.5)	na	ns	2.4 (1.1–5.3)	na	—
eaeA	33 (11.7)	—	—	—	—	2.4 (1.1–5.3)	ns	na	11.2 (5.1–24.9)
paa	77 (27.3)	0.6 (0.3–1.0)	19.5 (10.1–37.7)	4.5 (2.6–7.8)	—	na	na	ns	2.0 (1.1–3.9)
sepA	50 (17.7)	2.0 (1.1–3.9)	2.6 (1.4–4.9)	3.2 (1.7–5.9)	44.7 (9.8–205.0)	—	11.2 (5.1–24.9)	2.0 (1.1–3.9)	ns

Odds ratio (OR) for significant associations between the virulence genes (95% confidence interval in parenthesis); OR>1 represent positive associations, and OR<1 represent negative associations; —, no statistically significant association (p≥0.05); na, no results available (the OR could not be calculated because none of the isolates carried one of the combinations of virulence genes).

ns, no statistics were determined.

Table 4.

Virulence Genes and Phylogenetic Groups of Escherichia coli Isolates (n=344)

Virulence gene profile		Phylogenetic group, no. (%) of isolates
Gene	No.(%) of isolates	B1 (n=282)	B2 (n=24)	D (n=10)	U^a (n=28)
elt	5 (1.5)	—	—	5 (50.0)	—
fasA	68 (19.8)	62 (22.0)	—	—	6 (21.4)
astA, estB	1 (0.3)	1 (0.4)	—	—	—
astA, fasA	7 (2.0)	7 (2.5)	15 (62.5)	—	—
eaeA, fasA	2 (0.6)	—	—	—	2 (7.1)
elt, fasA	97 (28.2)	72 (25.5)	—	1 (10.0)	9 (32.1)
fasA, paa	1 (0.3)	1 (0.4)	—	—	—
fasA, sepA	9 (2.6)	9 (3.2)	—	—	—
astA, elt, fasA	20 (5.8)	20 (7.1)	—	—	—
astA, estA, fasA	1 (0.3)	1 (0.4)	—	—	—
astA, estB, fasA	9 (2.6)	9 (3.2)	9 (37.5)	—	—
astA, faeG, fasA	2 (0.6)	2 (0.7)	—	—	—
eaeA, elt, fasA	9 (2.6)	—	—	—	—
eaeA, fasA, paa	13 (3.8)	12 (4.3)	—	—	1 (3.6)
elt, estB, fasA	1 (0.3)	1 (0.4)	—	—	—
elt, fasA, fedA	4 (1.2)	—	—	4 (40.0)	—
astA, elt, estB, fasA	10 (2.9)	—	—	—	10 (35.7)
astA, elt, fasA, sepA	8 (2.3)	8 (2.8)	—	—	—
eaeA, fasA, paa, sepA	8 (2.3)	8 (2.8)	—	—	—
estB, fasA, fedA, paa	4 (1.2)	4 (1.4)	—	—	—
astA, elt, faeG, fasA, sepA	2 (0.6)	2 (0.7)	—	—	—
astA, estB, fasA, fedA, paa	20 (5.8)	20 (7.1)	—	—	—
elt, estB, faeG, fasA, sepA	1 (0.3)	1 (0.4)	—	—	—
astA, elt, estB, faeG, fasA, sepA	10 (2.9)	10 (3.5)	—	—	—
astA, elt, estB, fasA, fedA, paa	19 (5.5)	19 (6.7)	—	—	—
eaeA, elt, estA, fasA, fedA, paa	1 (0.3)	1 (0.4)	—	—	—
eaeA, elt, estB, fasA, paa, sepA	1 (0.3)	1 (0.4)	—	—	—
astA, eaeA, elt, fasA, fedA, paa, sepA	1 (0.3)	1 (0.4)	—	—	—
astA, eaeA, elt, estB, faeG, fasA, paa, sepA	1 (0.3)	1 (0.4)		—	—
astA, eaeA, elt, estB, fasA, fedA, paa, sepA	9 (2.6)	9 (3.2)		—	—

U strains that were not assigned to a phylogenetic group.

Phylogenetic groups

The majority of E. coli (82%) was classified into phylogenetic group B1. Twenty-four isolates (7%) belonged to phylogenetic group B2 and 10 isolates (3%) to group D. Twenty-eight isolates (8%) were negative to all three genetic markers and not assigned to a phylo-group.

Association between antimicrobial resistance phenotypes, resistance genes, virulence genes, and phylogenetic groups

Due to low numbers of the E. coli isolates in the other phylogroups, associations between resistance phenotypes, resistance genes, and virulence genes were analyzed only among the phylotype B1 isolates. The varied significance of associations between resistance phenotypes and virulence genes was revealed (Table 5). Overall, negative associations were more frequently observed than positive associations. The strongest positive associations were identified between fedA and gentamicin resistance.

Table 5.

Association Between Resistance Phenotype and Virulence Genes in Escherichia coli in Phylogenetic Group B1 (n=282)

	AMP		CHP		CIP		GEN		STR		SUL		TET		TRI
Virulence gene (n)	No. ^a	Association ^b	No.	Association	No.	Association	No.	Association	No.	Association	No.	Association	No.	Association	No.	Association
elt (146)	131	—^c	120	2.2 (1.3–3.8)	58	0.3 (0.2–0.5)	96	—	116	—	89	0.6 (0.3–0.9)	142	—	108	—
estA (2)	2	—	2	—	2	—	2	—	2	—	2	—	2	—	2	—
estB (76)	76	na^d	46	0.4 (0.2–0.7)	33	0.6 (0.3–1.0)	64	3.8 (1.9–7.5)	73	9.3 (2.8–30.7)	58	1.8 (1.0–3.4)	76	na	37	0.2 (0.1–0.3)
astA (110)	100	—	84	—	53	—	80	1.7 (1.0–2.9)	97	2.8 (1.4–5.5)	83	1.9 (1.1–3.3)	110	na	73	0.5 (0.3–0.8)
faeG (16)	14	—	7	0.2 (0.1–0.6)	5	—	13	—	12	—	11	—	16	—	16	na
fasA (281)	256	—	211	—	150	—	184	—	221	—	188	—	268	—	210	—
fedA (54)	54	na	34	0.5 (0.3–0.9)	21	0.5 (0.3–0.9)	53	39.2 (5.3–288.7)	53	18.5 (2.5–136.8)	40	—	54	—	15	0.1 (0.03–0.1)
eaeA (33)	23	0.15 (0.06–0.4)	15	0.2 (0.1–0.5)	18	—	16	0.5 (0.2–0.9)	21	0.4 (0.2–0.9)	21	—	24	0.04 (0.01–0.2)	7	0.06 (0.02–0.15)
paa (77)	67	—	39	0.2 (0.1–0.3)	29	0.4 (0.2–0.7)	58	1.9 (1.1–3.5)	64	—	51	—	68	0.2 (0.05–0.5)	21	0.03 (0.01–0.06)
sepA (50)	40	0.28 (0.12–0.66)	26	0.3 (0.1–0.5)	23	—	33	—	38	—	37	—	50	—	33	—

Only antimicrobial resistance phenotypes with a significant association (p<0.05) with the virulence genes are shown.

No., number of isolates resistant to corresponding antimicrobial agents and carrying the relevance virulence genes.

Odds ratio (OR) for significant associations between virulence gene and antimicrobial resistance (95% confidence interval in parenthesis). OR>1 represents positive associations, and OR<1 represents negative associations.

No significant associations (p≥0.05).

na, no results available (the OR could not be calculated because none of the isolates carried one of the combinations of virulence genes and resistance genes).

AMP, ampicillin; CHP, chloramphenicol; CIP, ciprofloxacin; ERY, erythromycin; GEN, gentamicin; STR, streptomycin, SUL, sulfamethoxazole, TET, tetracycline; TRI, trimethoprim.

The association between resistance genes and virulence genes were conducted (Table 6). The bla _TEM, catA, dfrA1, and dfrA12 genes show negative associations with all the corresponding virulence genes (p<0.05). In contrast, the catB, aadB, and sul3 genes exhibited only positive associations (p<0.05). The strongest positive associations were between gene pairs fedA/aadA2.

Table 6.

Association Between Antimicrobial Resistance-Encoding and Virulence Genes in Escherichia coli in Phylogenetic Group B1 (n=282)

	Association between the following antimicrobial resistance-encoding genes and virulence genes (no. of isolates, OR(95% interval)) ^a,b
Gene	bla_TEM	cmlA	catA	catB	aadB	aadA1	aadA2	strA-strB	sul1	sul2	sul3	tetA	tetB	dfrA1	dfrA10	dfrA12
elt	115,0.4 (0.2–0.7)	108,2.4 (1.4–3.9)	1,0.1 (0.01–0.8)	27,—	34,—	88,—	77,—	95,—	2,—	21,0.4 (0.2–0.7)	79,—	113,—	66,1.7 (1.1–2.8)	14,—	31,—	87,—
estA	2,—	2,—	0,—	1,—	0,—	1,—	2,—	2,—	0,—	0,—	2,—	2,—	0,—	0,—	2,na	1,—
estB	67,—	46,—	0,na	24,2.1 (1.2–3.8)	29,2.3 (1.3–4.0)	64,5.3 (2.7–10.5)	55,3.2 (1.8–5.8)	65,3.5 (1.8–7.1)	0,—	38,6.9 (3.8–12.7)	58,3.4 (1.8–6.1)	57,—	28,—	4,—	0,na	18,0.13 (0.1–0.2)
astA	90,—	74,—	0,na	32,2.0 (1.1–3.6)	30,—	80,2.6 (1.6–4.4)	70,2.2 (1.3–3.5)	88,2.5 (1.4–4.4)	0,—	35,2.3 (1.3–4.1)	77,2.6 (1.5–.2)	89,1.8 (1.0–3.2)	40,—	2,0.14 (0.03–0.6)	25,—	47,0.4 (0.2–0.6)
faeG	14,—	7,—	0,—	2,—	10,5.4 (1.9–15.4)	13,—	5,—	4,0.1 (0.04–0.4)	0,—	0,na	11,—	7,0.2 (0.1–0.7)	10,2.8 (1.0–7.8)	2,—	3,—	8,—
fasA	238,—	181,—	10,—	61,—	73,—	167,—	147,—	193,—	2,—	63,—	158,—	209,—	109,—	22,—	68,—	162,—
fedA	44—	34,—	0,—	23,3.7 (2.0–7.1)	19,—	52,25.5 (6.1–107.4)	52,36.4 (8.7–153.1)	53,32.7 (4.4–240.8)	0,—	30,7.1 (3.7–13.6)	40,2.6 (1.4–5.1)	44,—	7,0.2 (0.1–0.4)	2,—	1,0.05 (0.01–0.3)	3,0.03 (0.01–0.1)
eaeA	11,0.05 (0.02–0.1)	15,0.4 (0.2–0.9)	0,—	0,na	5,—	10,0.3 (0.12–0.6)	11,0.4 (0.2–0.9)	21,—	0,—	1,0.1 (0.01–0.7)	21,—	24,—	2,0.1 (0.02–0.4)	0,—	5,—	5,0.1 (0.04–0.3)
paa	55,0.3 (0.15–0.6)	38,0.4 (0.2–0.7)	1,—	23,1.9 (1.0–3.4)	24,—	52,—	52,2.4 (1.4–4.2)	64,2.8 (1.5–5.5)	0,—	30,3.2 (1.8–5.8)	50,—	58,—	9,0.14 (0.07–0.3)	2,0.25 (0.06–1.1)	6,0.2 (0.1–0.5)	7,0.03 (0.01–0.1)
sepA	30,0.17 (0.1– 0.3)	26,0.5 (0.3–1.0)	0,—	0,na	19,2.0 (1.1–3.9)	30,—	20,—	30,—	0,—	1,0.1 (0.01–0.4)	37,2.6 (1.3–5.1)	41,—	18,—	2,—	13,—	24,—

Numbers of isolates, odds ratio (OR) for significant associations between virulence gene and antimicrobial resistance (95% confidence interval in parenthesis). OR>1 represents positive associations, and OR<1 represents negative associations; —, no significant associations (p≥0.05); na, no results available (the OR could not be calculated because none of the isolates carried one of the combinations of virulence genes and resistance genes).

For resistance genes with significant associations (p<0.05), their associations are as follows (gene [positive/negative associations]): blaTEM(catB, aadB, aadA1, strAB, sul2, sul3, tetB/cmlA); cmlA(catB, aadA2, dfrA12/bla _TEM, aadB, sul2, dfrA10); catA(dfrA10/-); catB(bla _TEM , cmlA, aadA1, aadA2, strAB, tetA/sul3, tetB); aadB(bla _TEM ,aadA1, aadA2, sul2, sul3/cmlA, strAB, tetA, dfrA12); aadA1(bla _TEM , catB, aadB,aadA2, strAB, sul2, sul3/dfrA10); aadA2(cmlA, catB, aadB, aadA1, strAB, sul2, sul3/dfrA10, dfrA12); strAB(bla _TEM , catB, aadA1, aadA2, sul2, sul3, tetA/aadB); sul2(bla _TEM , aadB, aadA1, aadA2, strAB, sul3/cmlA, dfrA10, dfrA12); sul3(bla _TEM , aadB, aadA1, aadA2, strAB, sul2, tetB, dfrA1, dfrA12/catB); tetA(catB, strAB, dfrA10/aadB, tetB); tetB(bla _TEM , sul3, dfrA12/catB, tetA, dfrA10); dfrA1(sul3, dfrA10, dfrA12/-); dfrA10(catB, tetA, dfrA1, dfrA12/cmlA, aadA1, aadA2, sul3, tetB); and dfrA12(cmlA, aadA2, sul3, tetB, dfrA1, dfrA10/aadB, sul2).

The associations among virulence genes were analyzed (Table 3). The presence of eight virulence genes (including estB, elt, fedA, fasA, sepA, paa, astA, and eaeA) was significantly different (p<0.05), while estA and fasA exhibited no significant associations (p>0.05). The strongest associations were between fedA and estB.

Discussion

The main findings of this study were the widespread and diverse resistance and virulence genes among commensal E. coli in pigs. Resistance to tetracycline, ampicillin, and streptomycin was detected at high frequency, similar to previous studies in the strains from either diseased or healthy pigs in Canada (Boerlin et al., 2005) and in China (Boerlin et al., 2005; Wang et al., 2010). This most likely reflects the extensive use of such antimicrobials in swine production. Despite the prohibition of chloramphenicol for use in food animals, a high chloramphenicol-resistance rate was found. The explanation may be co-selection of chloramphenicol-resistance genes and other resistance genes by other antimicrobials. This phenomenon has been previously observed in other bacteria of animal origins (Bischoff et al., 2005; Chuanchuen et al., 2008) and suggested the efficient horizontal transfer of chloramphenicol-resistance determinants (Karczmarczyk et al., 2011).

In addition, more than 50% were resistant to ciprofloxacin, an antibiotic of choice for E. coli infection treatment. This could be an alarm for complications in treatment of E. coli infections by fluoroquinolones in the future.

Gene cassettes encoding resistance to aminoglycosides and trimethroprim were prevalent. These gene cassettes have been frequently found in class 1 integrons in different bacteria from various sources in many countries (Sunde and Norstrom, 2006; Wechsiri et al., 2011), indicating their common occurrence among class 1 integrons (Zhang et al., 2009). The dfrA12-aadA2 cassette array was predominant and formerly found in E. coli and other bacterial species of different sources (Zhang et al., 2009; Chuanchuen et al., 2010), suggesting more stable incorporation of this gene cassette within integron structures (Rosser and Young, 1999) and its important role in dissemination of trimethroprim and aminoglycoside resistance. Most class 1 integrons carrying dfrA12-aadA2 array were conjugally transferred to Salmonella, providing evidence that commensal E. coli serve as reservoirs of resistance genes potentially available for transfer among E. coli isolates and other bacteria. An incomplete sat gene cassette was also commonly identified, suggesting its localization on plasmid-borne class 1 integrons that were co-selected by different resistance genes present on the same plasmid.

A diversity of resistance genes was found. Their existence was related to individual resistance phenotypes, suggesting their regular expression. Most E. coli isolates carried multiple genes mediating the similar resistance phenotypes (e.g., dfrA1, dfrA10, and dfrA12 for trimethoprim resistance). Co-existence of several resistance genes encoding the identical resistance phenotype could be explained by the existence of resistance genes on different genetic elements. In this study, tetA was predominant among the tetracycline-resistant isolates, consistent with a previous study (Sabarinanth, 2011). In contrast, previous studies showed that tetB was most prevalent among the tetracycline-resistant commensal E. coli (Delsol et al., 2005; Diarrassouba et al., 2007). Regardless of the tet gene type, healthy pigs play a role as an accumulator of tetracycline-resistance determinants. Most of the ampicillin-resistant isolates in this study carried a bla _TEM gene that has been shown to dominate among the E. coli animal isolates (Brinas et al., 2002; Guerra et al., 2003). The reason is still unclear and is most likely linked to the type of β-lactams used. Among the sul genes tested, sul3 was most commonly found, consistent with a previous study in Canada (Rosengren et al., 2009). This finding differed from a previous study in Germany where the high prevalence of sul2 was observed (Schwaiger et al., 2010). Such discrepancy may be attributed to different sulphonamide type and geographic region.

While amino acid changes Ser-83-Leu and Asp-87-Asn observed in GyrA were previously reported (Guerra et al., 2003), another amino acid substitution Gln-94-Pro has never been described in quinolone-resistant E. coli. A novel amino acid substitution Ser-58-Ile was observed in ParC, but its contribution to fluoroquinolone resistance in these isolates is unclear.

A variety of virulence genes were found in E. coli in this collection. It was previously demonstrated that virulence gene profiles of E. coli from diarrheic and healthy piglets were not different (Schierack et al., 2006). Even though the pathogenicity of the E. coli isolates was not determined in this study, the data confirmed that the presence of virulence genes is not an absolute indicator of pathogenicity of a bacterial strain. In fact, the ability of virulence factors to trigger diseases may require additional factors (e.g., stress of weaning, lack of maternal antibodies, or dietary changes) (Fairbrother et al., 2005). Interestingly, the occurrence of fasA was very high (98.3%). This observation was uncommon and disagreed with previous studies (Boerlin et al., 2005; Schierack et al., 2006). A possible explanation may be related to different types of vaccines used in different areas (Chen et al., 2004).

The correlation between resistance phenotypes and virulence genes varied. Resistance to gentamicin and streptomycin was significantly associated with increased prevalence of certain virulence genes, suggesting linkage of resistance and virulence genes on plasmids. The strongest association was between gentamicin resistance and fedA. This could be explained by the strong positive association between fedA and two gentamicin resistance genes aadA1 and aadA2, implying their possible co-localization on the same genetic elements (e.g., plasmids, transposons). Co-existence of resistance and virulence genes on the same plasmid was previously shown in the pig isolates (e.g., pCG86 carrying streptomycin, tetracycline, and sulphonamide resistance genes and the enterotoxin genes elt and estA) (Mazaitis et al., 1981). In contrast, resistance to ampicillin, ciprofloxacin, and trimethoprim was significantly associated with reduced frequencies of several virulence genes, suggesting that the use of these antimicrobials does not select for the virulent factors in E. coli in this study. The possible explanation of such negative associations may be the existence of resistance and virulence genes on plasmids in the same incompatibility group (Boerlin et al., 2005).

For associations between resistance and virulence genes, the strong positive associations were detected among some resistance and virulence gene pairs (e.g., aadA, fedA; strA-strB, fedA), suggesting that antimicrobial use in pig farm could select for virulence genes. However, such an association was not observed in a previous study (Rosengren et al., 2009). This may be because the relationship between resistance and virulence genes appears to be strain specific and due to different antimicrobial use in different geographical regions.

Conversely, the negative correlations were observed between some resistance genes and virulence factors (e.g., tetA, faeG; tetB, fedA; and cmlA, eaeA). This may be explained by the presence of the same clonal isolates or the existence of the genes on plasmids in the same incompatibility groups. However, the genetic relatedness and plasmid was not analyzed in this study and is currently under investigation in our laboratory.

The triplex-PCR technique was used to analyze the E. coli phylo-groups. The discriminatory power was calculated to be 0.6582 by Simpson diversity indexes (Hunter and Gaston, 1988), similar to a previous study in the pig isolates (Carlos et al., 2010). While most commensal E. coli were assigned to sister groups A and B1 (Bingen et al., 1998), the majority of E. coli in this study belonged to phylogenetic group B1. The B1–E. coli isolates were commonly found among diseased pigs (Wang et al., 2010) and hospitalized patients (Johnson et al., 2002), although extraintestinal pathogenic human strains are mainly assigned to phylogroups B2 and D (Bert et al., 2010). Since E. coli of animal origin shared common characteristics with the human strains and serve as a possible source of risks to humans (Johnson et al., 2002; Carlos et al., 2010), the possibility that E. coli in nonpathogenic phylogenetic groups may adapt to more virulent strains could not be overlooked.

In conclusion, the results in this study confirmed the significant role of commensal E. coli as reservoirs of resistance determinants. The linkage between resistance and virulence genes supports the concerns that on farm antimicrobial use can select for virulence traits in pigs. Therefore, restrictive policies on antimicrobial use in food-animals and a surveillance program implemented to monitor evidence of an association between antimicrobial resistance and virulence features need in commensal E. coli isolates may be needed.

Footnotes

Acknowledgments

This work was supported by the 90^th Anniversary of Chulalongkorn University Fund (Ratchadaphisek Somphot Endowment Fund) and partly funded by Chulalongkorn University Veterinary Science Research Fund (grant RG7/2553). K.K.L. is a recipient of the Graduate Scholarship program for Faculty Members from Neighboring Countries, Chulalongkorn University. We thank Novatis (Thailand) Ltd. Animal Health Business Unit for providing the bacterial strain.

Disclosure Statement

No competing financial interests exist.

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