Related Antimicrobial Resistance Genes Detected in Different Bacterial Species Co-isolated from Swine Fecal Samples

Abstract

A potential factor leading to the spread of antimicrobial resistance (AR) in bacteria is the horizontal transfer of resistance genes between bacteria in animals or their environment. To investigate this, swine fecal samples were collected on-farm and cultured for Escherichia coli, Salmonella enterica, Campylobacter spp., and Enterococcus spp. which are all commonly found in swine. Forty-nine of the samples from which all four bacteria were recovered were selected yielding a total of 196 isolates for analysis. Isolates were tested for antimicrobial susceptibility followed by hybridization to a DNA microarray designed to detect 775 AR-related genes. E. coli and Salmonella isolated from the same fecal sample had the most AR genes in common among the four bacteria. Genes detected encoded resistance to aminoglycosides (aac(3), aadA1, aadB, and strAB), β-lactams (ampC, ampR, and bla _TEM), chloramphenicols (cat and floR), sulfanillic acid (sul1/sulI), tetracyclines (tet(A), tet(D), tet(C), tet(G), and tet(R)), and trimethoprim (dfrA1 and dfh). Campylobacter coli and Enterococcus isolated from the same sample frequently had tet(O) and aphA-3 genes detected in common. Almost half (47%) of E. coli and Salmonella isolated from the same fecal sample shared resistance genes at a significant level (χ ², p < 0.0000001). These data suggest that there may have been horizontal exchange of AR genes between these bacteria or there may be a common source of AR genes in the swine environment for E. coli and Salmonella.

Introduction

T he emergence of antimicrobial resistance (AR) in bacteria poses a challenge to both human and animal health (Walsh and Fanning, 2008; Wassenaar and Silley, 2008). Infections that are difficult to treat or untreatable increase with AR, particularly as resistance to multiple antimicrobials increases. Antimicrobial use has been identified as one of the driving factors behind the increase in AR, and use of antimicrobials in animals has been the subject of much debate (Aarestrup and Wegener, 1999; Wegener et al., 1999; van den Bogaard and Stobberingh, 2000; McDermott et al., 2002; Bywater et al., 2004; Walsh and Fanning, 2008). However, AR and even multidrug resistance (MDR: resistance to two or more antimicrobials) have been shown to develop, be maintained, and spread without exposure to selective pressures. In addition, the withdrawal of antimicrobial use has not always resulted in a corresponding reduction in AR (Luo et al., 2005; Zhang et al., 2006; Wassenaar and Silley, 2008). Further, there is evidence that the withdrawal of antibiotics used for growth in animal production has in some cases resulted in a decline in animal health with no observed improvement in human health or reduction of AR in human isolates (Phillips et al., 2004; Phillips, 2007; Wassenaar and Silley, 2008).

One problem that needs to be addressed is the potential for the development and amplification of AR genes via horizontal transfer of genetic elements encoding AR (Michael et al., 2004; Boerlin and Reid-Smith, 2008). This type of transfer may occur from relatively innocuous commensal strains where AR develops, and can then be transferred to pathogens resulting in an MDR pathogen. This can take place in a variety of environments, including on-farm environments where animals are treated with antimicrobials, thus selecting for AR bacteria that could be transmitted to humans via contaminated food products. However, studies supporting these hypotheses have relied solely on observations of phenotypic resistance to imply the amplification and transfer of AR, whereas other studies have identified only a few genes potentially transferred between donor and recipient bacteria (Aarestrup, 1999; Akwar et al., 2007; Frye and Fedorka-Cray, 2007; Aarestrup et al., 2008b; Jakobsen et al., 2010). Recent advances in gene detection technology, such as DNA microarrays, enable the identification of large numbers of AR genes in a single assay and can contribute to a better understanding of gene transfer (Chen et al., 2005; Perreten et al., 2005; Bruant et al., 2006; Frye et al., 2006, 2009; Ma et al., 2007; Batchelor et al., 2008; Zou et al., 2009).

In this study, these new methods were used to investigate AR genes in isolates from the on-farm environment. A previously described DNA microarray designed to detect 775 AR-associated genes was used to identify AR genes and genetic elements in bacteria isolated from animal samples (Frye et al., 2006, 2009). This microarray method was applied to bacteria isolated from swine feces collected on-farm during the Collaboration in Animal Health Food Safety and Epidemiology (CAHFSE) pilot study in swine (www.aphis.usda.gov/cahfse/index.htm). The CAHFSE study included three U.S. Department of Agriculture (USDA) agencies: the Agricultural Research Service (ARS), Food Safety Inspection Service, and the Animal Plant Health Inspection Service (APHIS). Fecal samples from healthy animals were collected from multiple sites in several states from 2003 to 2006. Samples were cultured for Escherichia coli, Salmonella enterica, Campylobacter spp., and Enterococcus spp. A set of these samples where all four bacteria were isolated was selected and analyzed for AR genes to determine if common AR genes were shared among different bacterial species from the same sample.

Materials and Methods

Swine farm sampling and feces collection

Isolates were obtained from the CAHFSE swine program conducted by USDA from 2003 to 2006, as described on the Web site www.aphis.usda.gov/cahfse/index.htm. Methods are described in detail at www.aphis.usda.gov/cahfse/methods/methods.htm. In brief, quarterly visits were conducted at 39 pork production sites from 2003 to 2004 and 48 sites from 2004 to 2006 in selected counties of IA, NC, MN, MO, and TX; not all sites were sampled each quarter. At each visit, 40 fresh individual fecal samples were collected from the floor of pens or lots containing market weight pigs (22 weeks of age and older). Samples were taken from a variety of pens or lots. Feces were collected only from portions of the droppings not in contact with the floor. Feces were placed in sterile plastic bags, packed with ice packs, and shipped overnight to ARS in Athens, Georgia, for culture of E. coli, Salmonella, Campylobacter spp., and Enterococcus spp.

Bacterial isolation and identification

Generic Escherichia coli

One-hundred-microliter aliquots of fecal dilutions (1:9 wt/vol, in phosphate-buffered saline [PBS, 0.1 M, pH 7.2]) were streaked for isolation onto CHROMagar EEC™ (Hardy Diagnostics, Santa Maria, CA) plates. The plates were incubated for 18–24 h at 42°C, after which colonies indicative of E. coli were selected.

Salmonella

Feces (1 g) were incubated in 10 mL of GN Hajna (Difco Laboratories, Detroit, MI) for 18–24 h at 37°C, and Tetrathionate broth (Difco Laboratories) for 40–48 h at 37°C. After initial enrichments, aliquots (100 μL) were transferred to 10 mL of Rappaport-Vassiliadis R10 broth (Difco Laboratories), which were incubated for 18–24 h at 37°C. Ten- microliter aliquots of Rappaport-Vassiliadis R10 broth were then streaked onto Xylose-Lysine-Tergitol-4 (Difco Laboratories) and Brilliant Green Sulfa (Difco Laboratories) agar. Plates were incubated for 18–24 h at 37°C. Isolated colonies characteristic of Salmonella were inoculated into triple sugar iron and lysine iron agar slants for biochemical confirmation. Presumptive positive isolates were serogrouped using serogroup-specific antisera (Difco Laboratories) and sent to National Veterinary Services Laboratories, APHIS, USDA (Ames, IA), for serovar determination.

Campylobacter spp

Fecal samples were diluted (1:9 wt/vol, in PBS) and 100 μL aliquots were inoculated onto Campy-Cefex agar plates (Stern et al., 1992) and into 1 mL Bolton Broth enrichment media in 24-well tissue culture plates (Falcon 353047; Becton Dickinson Labware, Franklin Lakes, NJ). Agar plates and enrichment broth were incubated for 36–48 h at 42°C under microaerobic conditions (5% O₂, 10% CO₂, and 85% N₂). Presumptive Campylobacter colonies were selected by observation of cellular morphology and motility using a wet mount under phase-contrast microscopy. Isolates were identified by species using a commercial multiplex PCR (BAX^® PCR; DuPont Qualicon, Wilmington, DE).

Enterococcus spp

One-hundred-microliter aliquots of fecal dilutions (1:9 wt/vol, in PBS) were inoculated into 24-well tissue culture plates (Becton Dickinson Labware) containing 1 mL of Enterococcosel broth (Becton Dickinson, Sparks, MD) per well. The plates were incubated for 18–24 h at 37°C, followed by streaking for isolation onto Enterococcosel agar (Becton Dickinson). Isolates were identified by species using a multiplex PCR (Jackson et al., 2004a).

Bacterial growth conditions and antimicrobial susceptibility testing

E. coli and Salmonella were grown in Luria Bertani (LB) media, on LB agar or blood agar plates at 37°C as indicated. Campylobacter coli were grown on Campy-cefex plates or in Bolton Broth and incubated at 42°C for 48 h under microaerobic conditions (5% O₂, 10% CO₂, and 85% N₂) in zip-top storage bags. Enterococcus spp. were grown in LB, brain heart infusion, or on blood agar plates at 37°C with standard methods. All bacteria were stored by addition of glycerol (10% final volume) to cells grown in liquid media and freezing at −80°C.

Susceptibility testing for E. coli, Salmonella, and Enterococcus spp. was performed using custom-made broth microdilution plates for the Sensititer™ system (TREK Diagnostic Systems, Inc., Westlake, OH) as described previously (www.ars.usda.gov/Main/docs.htm?docid6750&page = 2). Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards, NCCLS) guidelines for interpretation and recommended quality control organisms were used when available (NCCLS, 2002, 2003). Campylobacter isolates were tested following CLSI guidelines using the E-test (AB-Biodisk, Piscataway, NJ) or Sensititer (NCCLS, 2002, 2003). The antimicrobials tested and percentages of bacteria resistant to them are presented in Table 2.

DNA extraction and labeling

Genomic DNA from E. coli, Salmonella, and Enterococcus spp. isolates was extracted from 5 mL of overnight cultures grown in LB (Gram negative bacteria) or brain heart infusion (Gram positive bacteria) broth using the GenElute Bacterial Genomic DNA kit (Sigma, St. Louis, MO) as described previously (Frye et al., 2006). Camplyobacter genomic DNA was isolated from colonies collected from Campy-cefex plates using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) according to manufacturer's directions. DNA was labeled with Cye dye-labeled dCTP (Amersham, Piscataway, NJ) via random priming and extension with Klenow fragment (New England Biolabs, Beverly, MA), followed by purification with a Qiagen PCR clean up kit (Qiagen, Valencia, CA) as previously described (Porwollik et al., 2001).

DNA microarray design and construction

The array consists of probes designed to detect 775 AR-associated genes and was constructed as described previously (Frye et al., 2006, 2009; Zou et al., 2009). Controls included three positive control probes designed to detect bacterial 16S rDNA sequences (Liu et al., 2005), two Cy3- and Cy5-labeled lambda DNA controls, 12 buffer (50% dimethyl sulfoxide) only spots, and 130 empty (background) spots. Twelve probes were also synthesized in duplicate or triplicate as controls. All probes were synthesized (Operon Inc., Huntsville, AL), diluted in printing buffer (35 μM in 50% dimethyl sulfoxide), and arrayed in triplicate onto UltraGaps slides (Corning Inc., Life Sciences, Acton, MA) with an Omnigrid robot (Genemachines, San Carlos, CA) and post-processed as described previously (Porwollik et al., 2003; Frye et al., 2006).

Hybridization and scanning

Dye-labeled DNA was dried and resuspended in 80 μL of hybridization buffer (25% Formamide, 5 × SSC, 0.1% SDS, and 1% BSA), boiled for 5 min, and applied to the microarray under a LifterSlip (Erie Scientific, Portsmouth, NH). Hybridization was performed overnight in a hybridization chamber (Corning Inc., Life Sciences) submerged in a 42°C water bath in the dark. Protocols suggested by the manufacturer (Corning, Inc., Life Sciences) for hybridizations in formamide buffer were used for prehybridization, hybridization, and posthybridization wash processing (step 1: 2 × SSC, 0.1% SDS, 5 min at 42°C; step 2: 0.1× SSC, 0.1% SDS, 10 min at room temperature; step 3: 4× 0.1× SSC, 1 min at room temperature). Microarrays were scanned with a ScanArray Lite Laser scanner (PerkinElmer Life and Analytical Sciences, Waltham, MA) using ScanArray Express 3.0 software or with a GenePix 4100A (Molecular Devices, Sunnyvale, CA) and GenePix Pro software.

Hybridization analysis and scoring

Hybridizations were analyzed as previously described (Frye et al., 2006, 2009; Zou et al., 2009). Briefly, images were analyzed and quantified using ScanArray Express 1.1 software (PerkinElmer Life and Analytical Sciences). Hybridization signal intensities were measured in arbitrary intensity units by adaptive quantification mode followed by local background subtraction; the median values of the triplicate gene probes were recorded. Gene presence or absence was determined by setting a cutoff at two times the median intensity for all probes on the array. Genes with hybridization intensities greater than this cutoff were scored as detected (Frye et al., 2009; Zou et al., 2009). Hybridizations without detection of at least one 16S rDNA or with obvious hybridization anomalies (high background, streaks, spots, blotches, etc.) were considered failed and were repeated.

Data and statistical analysis

Genes detected in different bacteria species and samples were tabulated in Excel^® spreadsheets and totaled, and genes shared by different bacteria isolated from the same sample were determined. The χ ²-test was used to compare the predicted frequency of resistance genes detected in different bacteria from the same sample to the actual frequency observed. Pairs of isolates where <10 genes were detected in one isolate were excluded from analysis. The χ ²-test was considered significant at p < 0.0000001.

Cluster analysis of isolates with resistance genes detected

Relationships between isolates were determined by hierarchical cluster analysis using open source CLUSTER 3.0 with Euclidean distance for genes detected (Eisen et al., 1998; de Hoon et al., 2004). The dendrogram was viewed using Java TreeView version 1.1.4r3 (http://jtreeview.sourceforge.net) (Saldanha, 2004).

Plasmid replicon typing

Three panels of multiplex PCR were used to determine the presence of 18 plasmids commonly found in E. coli and Salmonella and to identify them based on incompatibility (Inc) groups. PCR primers and conditions were previously described (Carattoli et al., 2005; Lindsey et al., 2009).

Results

Bacteria recovered from swine fecal samples selected for analysis

A total of 7960 swine fecal samples were collected from 2003 to 2006 by the CAHFSE program. All of these samples were cultured for Salmonella and ∼40% (n = 3198) of the samples were also cultured for the other three bacteria. E. coli and Enterococcus spp. were isolated most often at 88.7% and 63.9% of samples, respectively. Campylobacter and particularly Salmonella were less frequently isolated at 61.0% and 9.1%, respectively. Phenotypic testing, including species determination, serovar determination, and antimicrobial susceptibility testing, was completed on all isolates (data available at www.aphis.usda.gov/cahfse/results/index.htm). Of the 3198 samples cultured for all four bacteria, 118 (3.7%) were positive for all four bacteria. These were screened for AR and 50 sample sets with the highest amount of resistances among their isolates were selected for microarray analysis. A Campylobacter isolate from one sample set could not be recovered from frozen stocks; therefore, that sample was eliminated from the study, leaving 49 samples with isolates of each of the four species for analysis (196 total) (Table 1).

Table 1.

Phenotypes of Isolates for Each Sample

Sample	Escherichia coli resistance(s)	Salmonella serotype	Salmonella resistance(s)	Campylobacter coli resistance(s)	Enterococcus spp.	Enterococcus resistance(s)
1	AmpSulTet	Derby	Tet	Tet	faecium	BacFlaTet
2	StrTet	Derby	None	CliTet	faecalis	BacEryLincTetTyl
3	AmpCepKanTet	Derby	KanTet	AziEryTet	mundtii	BacFlaLinc
4	SulTet	Derby	Kan	AziCliEryTet	faecalis	BacEryLincTet
5	AmpTet	Heidelberg	AmpKanStrTet	AziEryTet	casseliflavus	BacEryFlaLincStrTetTyl
6	SulTet	Infantis	ChlStrSulTet	Tet	faecalis	BacEryKanLincStrTetTyl
7	AmpStrTet	Derby	StrSulTet	AziCliEryTet	faecalis	BacEryLincTetTyl
8	Tet	Mbandaka	SulTetTri	None	faecalis	BacCipFlaLincTet
9	StrTet	Mbandaka	SulTetTri	None	faecium	BacFlaLincTet
10	StrTet	Derby	Tet	AziCliEryTet	casseliflavus	BacEryFlaLincTetTyl
11	AmoAmpFoxCepTetTio	Derby	Tet	AziCliEryTet	hirae	BacEryFlaLincNitSynTetTyl
12	Tet	Bovismorbificans	SulTet	Tet	faecalis	BacEryLincTetTyl
13	AmpKanSulTet	Typhimurium	AmpKanStrSulTet	AziEryTet	faecalis	BacEryLincStrTetTyl
14	Tet	Typhimurium	AmpKanStrSulTet	AziCliEryTet	faecalis	BacEryLincStrTetTyl
15	Tet	Typhimurium	AmpKanStrSulTet	AziCliEryTet	faecalis	BacLinc
16	SulTri	Typhimurium	AmpKanStrSulTet	Tet	faecalis	BacChlEryGenKanLincStrTetTyl
17	Tet	Typhimurium	AmpCepKanStrSulTet	AziEryTet	durans	FlaLincTet
18	AmpKanSulTet	Typhimurium	AmpKanStrSulTet	AziCliEryTet	faecalis	BacEryLincStrTetTyl
19	AmpKanStrSulTet	Derby	StrSulTet	AziEry	faecalis	BacChlEryKanLincStrTetTyl
20	Tet	Derby	StrSulTet	Tet	faecalis	BacEryKanLincStrTetTyl
21	StrTet	Newport	AmoAmpFoxCepChlStrSulTetTio	AziCliEryTet	Faecalis	BacEryLincTetTyl
22	AmpSulTet	Derby	KanTet	Tet	faecalis	BacEryLincTetTyl
23	KanStrSulTet	Derby	Tet	None	gallinarum	BacEryFlaKanLincStrTetTyl
24	Tet	Derby	AmoAmpFoxCepChlStrSulTetTriTio	AziCliEry	hirae	BacEryFlaKanLincPenStrSynTetTyl
25	AmpGenKanStrTet	Worthington	Tet	AziCliEryTet	faecalis	BacEryLincTetTyl
26	AmpGenKanStrTet	Typhimurium var. 5-	None	AziCliEryTet	faecalis	BacEryLincTetTyl
27	Tet	Heidelberg	AmoAmpFoxCepKanStrTetTio	AziCliEryTet	faecalis	BacEryLincTetTyl
28	AmpTet	Mbandaka	SulTetTri	AziEryTel	faecalis	EryLinNitStrTetTyl
29	AmpTet	Heidelberg	KanStrTet	AziEryTet	hirae	BacEryLincSynTetTyl
30	AmpStrTet	Mbandaka	SulTetTri	AziEryTet	hirae	BacEryFlaLincNitSynTetTyl
31	AmpCepSulTetTri	Mbandaka	SulTetTri	AziEryGenTelTet	hirae	BacEryFlaLincNitSynTetTyl
32	KanTet	Derby	StrSulTet	AziEry	faecalis	BacLincTet
33	StrSulTet	Typhimurium var. 5-	AmpChlStrSulTet	None	hirae	BacEryFlaLincNitPenSynTetTyl
34	StrTet	Typhimurium var. 5-	KanStrTet	AziEryTet	faecalis	BacChlEryLIncTet
35	AmpKanStrSulTet	Typhimurium var. 5-	AmpChlStrTet	AziEryTet	solitarius	FlaNit
36	Tet	Typhimurium var. 5-	AmpStrSulTet	AziCliEryTet	faecalis	BacLincTet
37	KanSulTet	Typhimurium var. 5-	AmpChlStrSulTet	AziCliEry	hirae	BacEryKanLincStrTetTyl
38	StrTet	Heidelberg	AmoAmpFoxCepKanStrTetTio	AziEryTet	faecalis	BacLincTet
39	AmpChlTetTri	Heidelberg	AmoAmpFoxCepKanStrTetTio	AziCliEryTet	faecium	BacCipFlaLincNitStrTet
40	AmpTet	Derby	StrSulTet	AziEryTel	durans	BacLincTet
41	StrTetTri	Mbandaka	TetTri	AziCliEryTet	faecalis	BacLinc
42	Tet	Derby	AmoAmpFoxCepGenStrSulTetTio	AziEry	solitarius	BacEryFlaLincNitTetTyl
43	Tet	Typhimurium var. 5-	AmpChlSulTet	Tet	hirae	FlaLincTet
44	AmpTet	Derby	StrSulTet	AziEryTet	faecalis	BacEryLincStrTetTyl
45	Tet	Derby	StrSulTet	AziCliEry	faecalis	BacEryLincStrTetTyl
46	AmpStrTet	Derby	AmoAmpFoxCepChlStrTetTio	CiNalTet	faecalis	BacChlEryGenKanLincStrTetTyl
47	AmpStrTet	Untypable	AmoAmpFoxCepChlGenStrSulTetTio	CiNal	faecalis	BacEryGenKanLincStrTetTyl
48	AmpStrTet	Derby	AmoAmpFoxCepChlGenStrSulTetTio	CiNal	hirae	FlaLincTet
49	Tet	Typhimurium var. 5-	AmpStrSulTet	AziCliEryTet	hirae	BacEryFlaLincNitPenSynTetTyl

Clinical and Laboratory Standards Institute breakpoints were used to determine resistance phenotype when available.

Gen, gentamicin; Kan, kanamycin; Str, streptomycin; Bac, bacitracin; Amc, amoxicillin-clavulanic acid; Amp, ampicillin; Cef, cephalothin; Cro, ceftriaxone; Cep, cefepime; Fox, cefoxitin; Pen, penicillin; Tio, ceftiofur; Chl, chloramphenicol; Fla, flavomycin; Van, vancomycin; Cli, clindamycin; Lin, lincomycin; Ery, erythromycin; Tyl, tylosin; Met, metronidazole; Nit, nitrofurantoin; Linc, lincomycin; Ci, ciprofloxacin; Nal, nalidixic acid; Syn, Synercid; Sul, sulfisoxazole; Tet, tetracycline; Tri, trimethoprim-sulfamethoxazole.

Phenotypic data for isolates of each bacterial species from the 49 samples were collected. None of the E. coli isolates were O157:H7 based upon colony morphology observed on CHROMagar. All Campylobacter isolates were assayed by Bax PCR and found to be C. coli (Table 1). The S. enterica isolate serovars were Derby (n = 19), Typhimurium (6), Typhimurium var. 5- (8), Heidelberg (5), Bovismorbificans (1), Newport (1), Infantis (1), Worthington (1), Mbandaka (6), and Untypable (1). Enterococcus species isolated included E. faecalis (28), E. durans (2), E. hirae (10), E. solitarius (2), E. casseliflavus (2), E. faecium (3), E. mundtii (1), and E. gallinarum (1) (Table 1).

Antimicrobial susceptibility of isolates

AR phenotypes for each isolate are shown in Table 1. Antimicrobials tested and total resistance found for each genus or species of bacteria are summarized in Table 2. Most of the isolates were resistant to one or more of the antimicrobials tested. Many of the E. coli and Salmonella isolates were resistant to aminoglycosides, β-lactams, folic acid pathway inhibitors, chloramphenicol, and tetracycline. None were resistant to members of the quinolone class of antimicrobials. For this study, resistance to more than one class of antimicrobial was considered MDR. The most common MDR E. coli detected were resistant to tetracycline and streptomycin (n = 6); ampicillin and tetracycline (n = 5); and ampicllin, streptomycin, and tetracycline (n = 5). Only two Salmonella isolates were susceptible to all antimicrobials tested (pan-susceptible); however, resistance was detected to all antimicrobials tested except the quinolones. Most of the Salmonella isolates (n = 41) were MDR. Salmonella Typhimurium was resistant to five or more antimicrobials more often than any other serovar, and resistance to eight or more antimicrobials occurred among serovars Newport, Derby, Heidelberg, and one untypable (Table 1). The C. coli isolates had the least resistance detected, and four isolates were susceptible to all antimicrobials tested (Table 1). The most common MDR phenotype for C. coli was detected in 15 isolates and included resistance to azithromycin, erythromycin, clindamycin, and tetracycline (Table 1). All Enterococcus spp. isolates were resistant to at least two antimicrobials tested, and one was resistant to 10 drugs. The most prevalent MDR phenotype detected was resistance to bacitracin, erythromycin, lincomycin, tetracycline, and tylosin, all of which were E. faecalis. The most extensively resistant isolate was resistant to bacitracin, erythromycin, flavomycin, kanamycin, lincomycin, penicillin, streptomycin, synercid, tetracycline, and tylosin (Table 1).

Table 2.

Totals of Antimicrobial Resistance for Each Bacteria

Antimicrobial class	Antimicrobial	Abbreviation	E. coli	Salmonella	C. coli	Enterococcus	Sum	% of tested ^a
Aminoglycoside	Gentamicin	Gen	2	3	1	3	9	4.6
	Kanamycin	Kan	10	15	NT	9	34	23.1
	Streptomycin	Str	18	31	NT	17	66	44.9
Beta-lactam	Amoxicillin/Clavulanic Acid	Amo	1	9	NT	NT	10	10.2
	Ampicillin	Amp	22	22	NT	NT	44	44.9
	Ceftriaxone	Axo	0	0	NT	NT	0	0.0
	Cephalothin	Cep	3	9	NT	NT	12	12.2
	Cefoxitin	Fox	1	7	NT	NT	8	8.2
	Penicillin	Pen	NT	NT	NT	3	3	6.1
	Ceftiofur	Tio	1	9	NT	NT	10	10.2
Folate pathway inhibitor	Sulfisoxazole	Sul	13	30	NT	NT	43	43.9
	Trimethoprim/Sulfamethoxazole	Tri	4	7	NT	NT	11	11.2
Ketolide	Telithromycin	Tel	NT	NT	3	NT	3	6.1
Lincosomide	Clindamycin	Cli	NT	NT	19	NT	19	38.8
	Lincomycin	Linc	NT	NT	NT	47	47	95.9
Macrolide	Azithromycin	Azi	NT	NT	34	NT	34	69.4
	Erythromycin	Ery	NT	NT	34	34	68	69.4
	Tylosin	Tyl	NT	NT	NT	32	32	65.3
Nitrofuran	Nitrofurantoin	Nit	NT	NT	NT	9	9	18.4
Phenicol	Chloramphenicol	Chl	1	10	0	4	15	7.7
Phosphoglycolipid	Flavomycin	Fla	NT	NT	NT	18	18	36.7
Polypeptide	Bacitracin	Bac	NT	NT	NT	43	43	87.8
Quinolone	Nalidixic acid	Nal	0	0	3	NT	3	2.0
Fluoroquinolone	Ciprofloxacin	Cip	0	0	3	2	5	2.6
Streptogramin	Synercid	Syn	NT	NT	NT	7	7	14.3
Tetracylcine	Tetracycline	Tet	48	46	35	45	174	88.8

Percent based only on bacteria tested with this antimicrobial.

NT, not tested for this bacteria.

AR genes detected in isolates

Hybridization data for all isolates are summarized in Table 3. A completed dataset is available in Supplementary Table S1 (Supplementary Data are available online at www.liebertonline.com/fpd). Overall, most isolates hybridized to multiple resistance gene probes and 16S rDNA-positive control probes. The 49 E. coli isolates had the most probe hybridizations with an average of 60 genes detected in each isolate. Aminoglycoside resistance genes detected and the number of isolates in which they were found included strAB (n = 17), aac(6) (n = 15), aadA1 (n = 18), and aadB (n = 16). A wide variety of β-lactam resistance genes were detected in the E. coli isolates, including cmy-2 (n = 5), tem (n = 14), sme-1 (n = 12), ampR (n = 18), bla2 (n = 15), and ampC (n = 33). Genes found in the isolates encoding resistance to folate pathway inhibitors included sulfisoxazole resistance genes sulI (n = 3) and sulII (n = 4), as well as trimethoprim resistance genes dfrA1 (n = 20) and dfh (n = 13). Chloramphenicol resistance genes identified were cat (n = 12) and floR (n = 6). Multiple isolates had tetracycline resistance genes detected that were dominated by tet(D) (n = 32), tet(A) (n = 31), and tet(R) (n = 21). MDR efflux pump-associated genes were found in the isolates including members of the marRABC (n = 32) regulatory system and acrA (n = 40) membrane transport system. Heavy metal resistance genes were detected in many isolates, including genes from the mer operon (merRTPCAD) in 19 of the isolates. Genes conferring resistance to sanitizers (quaternary ammonium compounds) qac were identified in 13 isolates. Genes often associated with mobilization of AR were found and included insA (n = 42), intI (n = 19), and tnpA (n = 23), and genes associated with conjugation in Inc HI1 plasmids (n = 19) were found in the isolate from sample 18.

Table 3.

Summary of Microarray Gene Probes with Positive Hybridizations to Isolates in the Samples Indicated ^a,b,c

Sample ID	Escherichia coli	Salmonella enterica	Campylobacter coli	Enterococcus spp.
1	acc aad str ampC ampR blaCMY blaTEM blaL2 blaSME mar mer qac sulII tet(A,B,C,D,R) ins int tnp dfrA1	aac blaL2 mar mer tet(A,C,D,R) ins int	cam-1 tet(O)	tet(M,O) ins tnp trans
2	str ampC blaSME-1 mar mer tet(R) ins int	mer	aphA-3 str ampC ampR cam-1 cfxA cat cme tet(O) dhf	aac aad str ampC ampR blaTEM-1 cfxA pre sme-1 cat linB ermB qac tet(A,D,M,S,O,W) ins int trans tnp dfh
3	aad ampC blaTEM mar tet(A,B,C,D,R) ins tnp dfrA1	aad aph3 ampC mar tet(A,B,C,D,R) tnp	aphA-3 cam-1 cml cme tet(O)	tnp
4	aac aad ampC ampR blaL2 cfxA blaSME blaTEM cat mar mer qac sulI tet(A,D,R) ins int tnp dhf	aad aph3 ampC blaSME mar mer tet(A,B,C,D,R)ins tpn	aphA-3 cam-1 cme tet(O)	pre ermB tet(M,O) tnp trans
5	ampC blaTEM mar mer tet(A,B,C,D,R) ins tnp dfrA1	aad str blaTEM cat mar qac tet(A,B,C,D,R) tnp dfrA1	cme tet(O)	ermB tnp trans
6	aac aadaphA-3str ampC ampR blaTEM blaSME mar mer sulII tet(A,B,C,D,M,R,W) ins tnp dfh	aac aad str flo mar mer sulII tet(A,D) tnp dfrA1	blaL2 cam-1 cme tet(O)	aad aphA-3 ermB trans
7	ND	str blaL2 cat tet(A,R) ins	cme tet(O)	ermB tet(M) tnpA trans
8	aad strampC ampR blaL2 cfxA blaSME cat mar mer qac tet(A,D,R) ins int	aad str ampC ampR blaL2 cfxA cat flo mar mer qac sulI tet(A,R) ins int tnp dhf	aphA-3 str cam-1 cme tet(O)	pre tet(M) tnpA trans
9	str ampC mar tet(A,B,R) ins	ND	aphA-3 tet(O)	aac pre linB tet(M,O,W)ins tnp trans
10	aac aad str ampC ampR blaL2 cfxA blaSME cat mar mer qac tet(A,B,C,R)ins int tnp dhf	aad mar tet(A,B,C,D,R) ins tnp	aphA-3 cam-1 cme tet(A,O)	ermB tet(S) tnp trans
11	aac ampC cfxA blaCMY mar mer tet(A,B,C,D,R)ins int tnp dfrA1	mar tet(A,B,C,D,R)	aphA-3 cam-1 cme tet(O)	aac str blaL2 cat cml qac tet(A,M,O) ins int tnp trans
12	blaL2 tet(A,B,C,D,R) ins	aadA tet(A,B,D,R)	aphA-3 cam-1 cme tet(O)	ermB tet(O) trans
13	aad ampC ampR cfxA blaSMEmar mer qac tet(A,B,D) ins int tnp dfrA1	aad ampC blaTEM cat cml mar mer qac tet(A,B,C,D,R)ins int tnp dfrA1	cme tet(O)	aac strB blaL2 pre cat cml ermB qac tet(A,M,O)ins int tnp trans
14	ins	aac aad aph3 ampC ampR blaTEM cfxA blaSME cat flo sulI mar qac sulI tet(A,R) ins int tnp dfh	cme tet(M,O)	pre linB ermB tet(M,O,W) tnp trans
15	acc aad aph3ampC ampR cat mar qac tet(A,B,C,D,R) ins int tnp dfrA1	aac aad aph3 blaTEM mar mer qac sulI tet(A,R) ins int tnp trans dfrA1	aphA-3 cme tet(O)	aad ampC blaL2 qac sulI tet(A,R) ins int
16	aac aad str ampC ampR cfxA blaCMY blaL2 blaSME mar mer qac sulII tet(A,D,R) ins int tnp dfrA1 dhf	aac aad aph3 str ampC ampR blaL2 cfxA blaSME blaTEM mar mer qac sulI tet(A,R)ins int tnp dhf	ND	acc str pre cat qac ermB tet(M,O,S,W) ins int tnp trans
17	mar tet(A,B,C,D,R) ins tnp	acc aad aphA-3 blaTEM mar mer qac sulI tet(A,R) ins int tnp	aac aad aphA-3 str blaL2 blaTEM cam-1 pre cat flo cme qac tet(A,M,O,S) ins int tnp	aac pre linB tet(M,O,W) tnp trans
18	aac aad aph3 str ampC blaTEM cat cml mar mer qac tet(A,B,C,D,R) ins int tnp dfrA1	aac aad str blaTEM blaSME mar mer qac sulI tet(A,R) ins int tnp	aphA-3 cam-1 cme tet(O)	ermB tet(O) tnp trans
19	aac aph3 str ampC blaTEM cfxA blaCMY cml mar mer qac tet(A,B,C,D,M,O,R) ins int tnp dfrA1	str blaL2	cme	aac aad aphA-3 ampCblaL2 pre flo ermB tet(O) tnp
20	tet(A,C,D) ins int tnp dfrA1	ND	tet(O)	aac aad aphA-3 pre linB ermB qac tet(M,O)ins int tnp trans
21	str ampC mar tet(A) ins tnp	aad str ampC blaCMY flo mar mer sulI sulII tet(A,R) tnp dfrA1	aphA-3 tet(O)	ermB tnp trans
22	aad ins	mar tet(A,B,C,D,R)	aac aad aphA-3 str blaL2 blaTEM cam-1 cfxA pre cat flo cme qac tet(A,M,O,S) ins int tnp	aad aphA-3 pre ermB tet(O) tnp trans
23	aph3 str ampC mar tet(C) ins	tet(A,B,C,D) ins	blaL2 cam-1 blaTEM cat tet(O)	aac aad aphA-3 str ampR blaCMY blaSME cat ermB qac tet(M,O,S,W) ins tnp trans
24	int tnp	aad str flo mar mer qac sulI sulII tet(A,R)ins int tnp dfr1	cam-1 cme tet(O)	aac aphA-3 pre cat linB ermB qactet(A,M) int tnp trans
25	aad aph3 str ampC blaTEM mar mer tet(A,B,C,D,M,O,R) ins int tnp tnp dfrA1	ND	aphA-3 sulII tet(O)	pre ermB
26	str blaTEM mar tet(A,C,D) ins int tnp	ND	acc aad ampC cam-1 cme sulII tet(O) ins int tnp trans	aac
27	ampC mar tet(A,B,D,R) ins int tnp	aac aad str ampC ampR blaL2 blaCMY blaTEM blaSHV cat flo mar mer qac sulI tet(A,B,C,D,R) ins int tnp dfrA1 dhf	acc cam-1 tet(O)	pre linB ermB tet(M,O) tnp trans
28	blaTEM tet(C,O,R) ins	aad mar qac sulI tet(A,R) int tnp dfrA1	ND	blaL2 pre linB tet(M,O) trans
29	ampC ins	aac aad str cfxA mar mer qac tet(A,B,C,D,R) tnp dfrA1	tet(O)	aac pre blaTEM ermB qactet(M,O) tnp trans
30	aac str ampC blaTEM mar tet(A,B,C,D,R) ins tnp dfrA1	ND	cam-1	blaL2
31	ins	aad mar qac sulI tet(A,R) int tnp dfrA1	tet(O)	aac blaL2 pre cat qac ermB sulI tet(A) trans
32	aac aad str ampC ampR blaL2 cfxA cat flo mar mer qac tet(A,D,R) ins int tnp dhf	aac aad str ampC ampR blaL2 cat flo mar mer qac sulI tet(A,D,R) ins int tnp dhf	aphA-3 cam-1 pre cme tet(O)	aac pre linB tet(M,O,W)tnp trans
33	aad str blaL2 mar qac sulI tet(A) ins tnp dfrA1	aad str blaPER mar flo qac sulI tet(A,R) int	cam-1 cme tet(O)	aac pre cat ermB qac tet(A,M,O,S,W) int tnp trans
34	aad mar tet(A,D,R)	aph3 tet(C,R) ins tnp dfrA1	aad aphA-3 cam-1 blaTEM cme tet(D,R) int dhf	pre ermB tet(O)
35	aac aad str ampR blaL2 blaTEM blaSME cat cml flomar mer qac sulI tet(A,D,R) ins int tnp dfrA1 dhf	aac aad str ampR blaL2 blaSME cat flo mar mer qac sulI tet(A,D,R) ins int tnp dhf	cme tet(O)	blaL2 cat tet(M,O) int tnp trans
36	aac aad str ampC ampR cat mar mer qactet(A,D,R) ins int tnp dfrA1 dhf	aad strA str ampRblaL2 blaTEM mar mer qac sulI tet(A,D,R) ins tnp	ND	aad blaL2 tet(A)
37	aac aad aph3 str ampC ampR blaL2 cfxA blaCMY blaSME cat flo mar mer qac sulII tet(A,B,C,D,R) ins int tnp dfh	aad flo qac	cam-1 cme tet(O) tnp	ermB trans
38	aac aadstr ampC ampR blaL2 cfxA cat mar mer qac tet(A,B,D,M,O,W) ins int tnp dfrA1 dhf	aac aad str ampR blaL2 blaCMY tet(A,B,D,R) ins int tnp dfrA1 dhf	aphA-3 cam-1 cme tet(C,O)	aad tet(M,O,W) ins tnp trans
39	aad str ampC ampR blaTEM catmar mer tet(A,B,D) ins tnp dfrA1	add str ampC ampR blaL2 blaCMY mar mer qac tet(A,B,C,D,R) ins int tnp dfrA1 dfh	str blaTEM cam-1 cfxA cat cme tet(O) int tnp	pre tet(M,O) ins tnp trans
40	aad ampC blaL2 blaTEM martet(A,B,C,D,R)ins tnp dfrA1	aad str ampR blaL2 mar mer qac sulI tet(A,R) insint	blaL2	acc aad str cat flo vanC ermB tet(M,O,R) ins int tnp
41	dfrV	aad str mar mer qac tet(A) int tnp dfrA1	aphA-3 cam-1 cme tet(O)	aac aad blaL2 tet(C) tnp dfrA1
42	acc aad ampC	aad str sulII tet(A,R) tnp dfrA1	cam-1 cme tet(O) ins	ND
43	aad tet(C.D,R) ins	aad flo mar qac sul1 tet(A) int	aphA-3 cam-1 cme tet(O)	pre cat tet(M,O) tnp trans
44	aac aad str ampC ampR blaL2 blaTEM cfxA blaSME cat flo mar mer qactet(A,C,R) ins int tnp dfrA1 dhf	aac aad str ampC ampR blaL2 cfxA blaSME cat flo mar mer qac sul1 sulI tet(A,D,R) ins int tnp dhf	aphA-3	blaL2 ermB tet(O)
45	aac aad str ampC ampR blaL2 cfxA blaSME cat flo mar mer qac tet(A,D,R) ins int tnp dfrA1 dhf	aac aad str ampC ampR blaL2 cat mar mer qac sulI tet(A,D,R) ins int tnp dfrA1 dhf	aad cam-1 cme tet(O) tnp	ermB tet(M)
46	ND	ND	aad str cam-1 blaSME cat cme tet(O) int tnp dhf	aphA-3 cat ermB
47	str ampC ampR blaTEM pre blaSME mar mer qactet(A)ins tnp	aac aad str ampC ampR blaL2 cfxA blaSME cat flo mar mer qac tet(A) ins int tnp dhf	cme tet(O)	ND
48	aac aad strampC ampR blaL2 cfxA cat mar mer tet(A,B,C,D,R) ins int dhf	aad str ampC ampR blaL2 blaCMY flo mar mer qac sulI sulII tet(A,R) ins tnp dfrA1 dhf	cat cam-1 cme	ND
49	mar int	aac aad str ampC ampR bla2 blaTEM cam-1 cat mar mer qac sulI tet(A,D,R) ins int tnp dfrA1 dhf	blaL2	trans

Genes and gene families with multiple probes are summarized and presented only once; ND indicates that none of the gene probes in this table were detected in that isolate (full hybridization data are available in Supplementary Table S1).

Probes with positive hybridizations to more than one isolate from the sample are indicated in bold.

Shaded samples hybridized to the same gene probes at a significant level as determined by the χ ²-test (p < 0.0000001).

Salmonella isolates also had a large number of positive probe hybridizations with an average of 56 genes detected per isolate. The most numerous aminoglycoside resistance genes detected in the Salmonella isolates were from the family of aadA genes followed by the str family. Genes detected and number of isolates in which they had been found were aadA1 (n = 30), aadA1b (n = 20), aadA2 (n = 20), aadB (n = 17), and strAB (n = 22). β-lactam resistance genes were detected and included blaL2 (n = 18), ampR (n = 15), ampC (n = 11), per-2 (n = 12), and tem (n = 10). Sulfamethoxazole and sulfisoxazole resistance genes detected most often in the Salmonella isolates were the highly homologous sul1 (n = 16) and sulI (n = 20) genes, whereas trimethoprim resistance genes detected were dfrA1 in 18 isolates and dfh in 13 isolates. Chloramphenicol resistance genes floR (n = 10) and cat4 (n = 9) were also detected. Tetracycline resistance genes found in most isolates included tet(A) (n = 22), tet(R) (n = 17), tet(C) (n = 13), and tet(D) (n = 21). Heavy metal resistance genes were also detected in several of the isolates with at least one mer gene detected in 21 of the isolates. Genes encoding resistance to sanitizers such as qacE were detected in 23 isolates. Many genes often mobilized with AR were also detected the most numerous were the transposase gene tnpAIS26 (n = 30) and the class I integrase gene intI1 (n = 16).

C. coli isolates had the lowest number of hybridizations to AR gene probes with an average of 17 genes detected per isolate. Over half of the C. coli isolates had aminoglycoside resistance genes detected and included aph-A3 (n = 19) and several close homologs to this gene (n = 24). The endogenous cam-1 β-lactamase gene was found in 30 of the isolates. Only three isolates had chloramphenicol resistance genes detected including floR, cat, and cmlA5. The cmeB and cmeC efflux pump genes were detected in 25 and 30 isolates, respectively. The tet(O) tetracycline resistance gene was found in a large number of the C. coli isolates (39), with just a few hybridizations to tet(A), tet(M), tet(R), and tet(S). Very few mobilization associated genes were found in the C. coli isolates.

The Enterococcus genera isolates had a moderate number of hybridizations to the gene probes with an average of 23 genes detected per isolate. The most prominent aminoglycoside resistance genes detected were acc(3) (n = 10) and aphA-3 (n = 7). Several β-lactam resistance genes were also detected including the blaL2 gene (n = 11) and the type-II SCCmec (methicillin resistance) cassette-associated gene pre (n = 22). The linB, lincomycin resistance gene, was detected in six isolates. The macrolide resistance gene most often found was the ermB gene, which was detected in 26 enterococcus isolates. Chloramphenicol resistance genes identified included the cat gene (n = 5). Several tetracycline resistance genes were detected, including tet(O) (n = 27), tet(M) (n = 23), tet(W) (n = 10), and tet(A) (n = 6). Some vancomycin resistance genes detected, including vanC (n = 1) and vanH (n = 4); additionally, a gene encoding resistance to the glycopeptide, bleomycin, was detected in eight isolates. Efflux pump genes were detected, including one associated with the type-II SCCmec cassette that was found in 22 isolates. Sanitizer resistance genes were detected, including the qacE gene, in eight isolates. Finally, multiple transposon genes often mobilized with resistance genes were detected, including tpnA (n = 28), a Tn1546 homolog, and trans (n = 35), another transposon related gene.

Genes detected in different bacterial species isolated from the same sample

To understand the distribution of resistance genes detected in the isolates, genes detected in the bacteria were compared to determine which genes were shared by the different bacteria isolated from the same fecal sample. Predominant genes detected are summarized in Table 3, with shared genes indicated by bold font and samples that share genes at a significant level by the χ ²-test indicated by shading.

E. coli and Salmonella had the most genes detected in common from the same sample at 1048, with 224 genes detected at least once in both Salmonella and E. coli isolated from the same sample. Aminoglycoside resistance genes were some of these, including aac(3) (shared in five samples), the family of aadA1 homologs (12 samples), aadB (8 samples), and strAB genes (10 samples). β-lactam resistance genes were found in common, the most numerous of which were ampC (8 samples) and its regulator, ampR (11 samples), blaL2 (10 samples), and others included class A β-lactamases, bla _SME-1 (five samples), bla _PER2 (five samples), and bla _TEM (two samples). Sulfamethoxazole and sulfisoxazole resistance genes were also shared, including the highly similar sul1 (one sample) and sulI (two samples) genes, as well as sulII (one sample). The most often shared trimethoprim resistance genes were dfrA1 (six samples) and dfh (eight samples). Chloramphenicol resistance genes were also detected in pairs of E. coli and Salmonella, including the cat gene in six samples and the floR gene in four samples. Tetracycline resistance genes were shared quite often and included tet(A) (17 samples), tet(D) (17 samples), tet(C) (six samples), and tet(R) (eight samples). Several members of heavy metal transport systems were also detected, including members of the mer family, the most common being merT, where it was detected in 13 pairs of isolates from the same sample. Quaternary ammonium resistance genes, qacE, were also detected in both E. coli and Salmonella from 8 samples. Genes associated with the transfer of resistance were detected in common, including insA (15 samples), intI1 (five samples), and tnpAIS26 (14 samples). For almost half of the samples (23), the correlation between genes detected in E. coli and Salmonella was determined to be significant (Tables 3 and 4, shaded samples). The most prevalent gene probes shared by pairs of E. coli and Salmonella isolated from each sample are summarized in Table 4 (complete data available in Supplementary Table S1).

Table 4.

Summary of Positive Microarray Gene Probe Hybridizations Shared by Escherichia coli and Salmonella enterica ^a,b

Sample ID	Gene probe hybridizations
1	aac blaL2 mar mer tet(A,C,D,R) ins int
2	mer
3	aad ampC mar tet(A,B,C,D,R) tnp
4	aad ampC mar mer tet(A,D,R) ins tpn
5	blaTEM mar tet(A,B,C,D,R) tnp dfrA1
6	aac aad str mar mer sulII tet(A,D) tnp
7	ND
8	aad str ampC ampR blaL2 cfxA cat mar mer qac tet(A,R) ins int
9	ND
10	aad mar tet(A,B,C,R) ins tnp
11	mar tet(A,B,C,D,R)
12	tet(A,B,D,R)
13	aad ampC mar mer qac tet(A,B,D) ins int tnp dfrA1
14	ins
15	aac aad aph3 mar qac tet(A,R) ins int tnp dfrA1
16	aac aad str ampC ampR blaL2 cfxA blaSME mar mer qac sulI tet(A,R) ins int tnp dhf
17	mar qac tet(A,R) ins tnp
18	aac aad str blaTEM mer qac tet(A,R) ins int tnp
19	str
20	ND
21	str ampC mar tet(A) tnp
22	ND
23	tet(C) ins
24	int tnp
25	ND
26	ND
27	ampC mar tet(A,B,D,R) ins int tnp
28	tet(R)
29	ND
30	ND
31	ND
32	aac aad str ampC ampR blaL2 cat flo mar mer qac tet(A,D,R) ins int tnp dhf
33	aad str mar qac sulI tet(A)
34	tet(R)
35	aac aad str ampR blaL2 blaSME cat flo mar mer qac sulI tet(A,D,R) ins int tnp dhf
36	aad strA str ampR blaL2 mar mer qac tet(A,D,R) ins tnp
37	flo qac
38	aac aad str ampR blaL2 tet(A,B,D) ins int tnp dfrA1 dhf
39	add str ampC ampR mar mer tet(A,B,D) ins tnp dfrA1
40	aad blaL2 mar tet(A,R) ins
41	ND
42	aad
43	aad
44	aac aad str ampC ampR blaL2 cfxA blaSME cat flo mar mer qac tet(A,R) ins int tnp dhf
45	aac aad str ampC ampR blaL2 cat mar mer qac tet(A,D,R) ins int tnp dfrA1 dhf
46	ND
47	str ampC ampR blaSME mar mer qac tet(A) ins tnp
48	aad str ampC ampR blaL2 mar mer tet(A,R) ins dhf
49	mar int

Genes and gene families with multiple probes are summarized and presented only once; ND indicates that none of the gene probes in this table were shared in isolates from this sample (full hybridization data are available in Supplementary Table S1).

Shaded samples hybridized to the same gene probes at a significant level as determined by the χ ²-test (p < 0.0000001).

The next most frequent bacterial species isolated from the same sample to share genes was Salmonella and Enterococcus at n = 125, with 64 probes shared by at least one pair of bacteria from the same sample. Several aminoglycoside resistance genes were detected in both of these bacteria from the same sample, including aadA1 (two samples), aadA2 (three samples), aadB (one sample), and strB (one sample). The blaL2 gene was detected in four Salmonella and Enterococcus sample pairs and was the only β-lactamase gene detected in common. The tetracycline resistance gene probes tet(A) (two samples) and tet(R) (one sample) were also detected. The quaternary ammonium resistance gene probe qacE was also detected in both isolates from five samples. The transposase gene probe insA also had positive hybridizations with seven pairs of Salmonella and Enterococcus. One sample (ID number 15) had genes shared at a significant level between Enterococcus and Salmonella, each of which also shared genes at a significant level with the E. coli isolated from that sample (Table 3).

A similar number of E. coli and Enterococcus pairs from the same sample had shared genes detected at n = 117, with 71 shared by at least one pair. Some of these included aminoglycoside resistance gene probes aadA1 (one sample), aadA2 (two samples), aphA3 (one sample), and strB (two samples); β-lactamase resistance gene probes ampC (two samples), blaL2 (two samples), bla _SME-1 (one sample); quaternary ammonium resistance gene probes qacE (two samples), qacF (one sample), and qacH (two samples); tetracycline resistance gene probes included tet(A) (two samples), tet(M) (one sample), tet(O) (three samples), tet(R) (one sample), and tet(W) (one sample); transfer-related gene probes included insA (five samples), intI1 (two samples), and transB (one sample). In addition, Salmonella, E. coli, and Enterococcus also had a few samples where all three bacteria shared a positive hybridization to the same probe (n = 62 for 33 probes). These included aadA1 (one sample), aadA2 (two samples), aadB (one sample), strAB (one sample), blaL2 (two samples), qacE (two samples), qacF (one sample), qacH (two samples), tet(A) (two samples), and insA (four samples).

The remaining pairs of bacteria had only a few genes found to be common, and all of these were pairs with Campylobacter, which had the lowest number of genes detected in the study. The total for Campylobacter and Enterococcus was 52, Campylobacter and Salmonella were 31, and Campylobacter and E. coli were 28. The tet(O) gene probe alone stood out as being shared by the Campylobacter with other bacteria isolated from the same sample in this study. The tet(O) gene was detected in 21 pairs of Campylobacter and Enterococcus and three pairs of Campylobacter and E. coli. Genes found in common with three or more bacteria isolated from the same sample were rare at 41 occurrences where three species shared the same hybridization. Some genes encoding resistance that fell into these categories were aadA1, aadA2, strB, blaL2, merR, qacE, tet(A), and tet(O). None of the probes hybridized to all four species from the same sample. None of the other pairs of isolates had genes in common from the same sample detected at a significant level.

Cluster analysis of isolates determined by resistance genes detected

The isolates in the study were grouped using cluster analysis based on presence or absence of resistance genes in each isolate. No isolates from the same sample grouped together, and almost all of the isolates grouped more closely to members of the same genus or species of bacteria than they did to different bacteria regardless of sample source (data presented in Supplementary Fig. S1). This was observed even when clustering analysis was done only on E. coli and Salmonella data or only on data from these bacteria with a significant number of genes shared (Supplementary Figs. S2 and S3). An exception to this was the E. coli isolate from sample 18 and the Salmonella isolate from sample 13 that grouped together and shared 94 out of 107 (87.9%) resistance genes detected (Table 3 and complete dataset in Supplementary Table S1). The remaining isolates of different bacteria that grouped loosely together by cluster analysis did so due to very few genes (<5) being detected in all of these isolates (Supplementary Figs. S1–S3, Table 3, and Supplementary Table S1). The genes detected in the isolates were also clustered. Probes for genes that shared sequence homology grouped together such as those for closely related alleles (e.g.bla _CMY-2 and bla _CMY-13). Genes associated with similar functions or pathways (e.g., mercury transport and conjugation) and genes often found in the same genetic element (e.g., integron and cassettes genes) also grouped loosely together (Supplementary Fig. S3).

Plasmid replicons detected

Plasmid replicon typing of E. coli and Salmonella isolates detected several replicons often associated with these species. Salmonella isolates had replicons detected that included IncA/C, B/0, FIA, FIB, FIIA, F, HI1, and P. E. coli isolates had IncB/O, FIC, FIIA, F, HI1, and Y plasmid replicons detected (Table 5). Isolates of E. coli and Salmonella from five samples had plasmid replicons detected in common. Sample 18 had the FIB replicon detected in both the E. coli and Salmonella isolates, and samples 36, 37, 44, and 45 had the IncF replicon detected in isolates of both species.

Table 5.

Plasmid Replicons Detected in Escherichia coli and Salmonella enterica Isolates

Black block indicates replicon detected; IncX, N, W, HI2, K, L/M, and T were not detected in any isolates.

Discussion

This study investigated genetic elements associated with AR shared by bacteria isolated from the same swine fecal sample. Samples were not randomly selected for this study and were chosen when E. coli, S. enterica, C. coli, and Enterococcus spp. were all successfully isolated, and were then selected to obtain the maximum number of AR phenotypes possible. Despite the selection criteria for the most resistances, overall the other phenotypic data are very similar to that found by the entire CAHFSE study (data available at www.aphis.usda.gov/cahfse/results/index.htm) and other surveillance programs in the United States such as the National Antimicrobial Resistance Monitoring System (NARMS) and the National Animal Health Monitoring System (NAHMS) as well as data published in on-farm studies by other researchers (Gebreyes et al., 2000, 2004, 2005; Gebreyes and Altier, 2002; Jackson et al., 2004a, 2005). AR phenotypes detected were also comparable to national slaughter surveillance results (NARMS) and other published reports (Jackson et al., 2004b; Gebreyes et al., 2005; Akwar et al., 2007; Thakur et al., 2007; Aarestrup et al., 2008a; Varga et al., 2008). These included high numbers of tetracycline-resistant, ACSSuT+ (AmpChlStrSulTet; the well-recognized penta-resistance profile)-resistant, and ceftiofur-resistant isolates of Salmonella; macrolide (AziEry) resistance in C. coli; and bacitracin, linconmycin, and macrolide (EryTyl) resistance in Enterococcus spp.

AR genes in the isolates were detected using a microarray allowing high-density analysis of each isolate with hundreds of AR gene probes (Frye et al., 2006, 2009; Zou et al., 2009). The genes detected were similar to those previously identified in swine isolates by other groups using PCR and sequencing methods as well as a few microarray studies (Gebreyes and Altier, 2002; Jackson et al., 2004b; Boerlin et al., 2005; Ma et al., 2007; Kozak et al., 2009). For example, E. coli isolates in the current study had positive hybridizations to probes for members of the aac, aad, aph, and str families of aminoglycoside resistance genes, including aac(3), aac(6), and aph3, which encode kanamycin resistance, and aadA1 and strA-strB, which encode streptomycin resistance. Many studies have established that these genes are often found in aminoglycoside-resistant E. coli isolated from pork, swine, or the swine environment (Sandvang and Aarestrup, 2000; Rosengren et al., 2009). β-Lactam resistance gene probes with positive hybridizations to the E. coli isolates included bla _TEM, ampC, and bla _CMY homologs, which is also similar to previous reports (Chen et al., 2005; Heider et al., 2009). One of the most often detected groups of genes encoded tetracycline resistance and included homologs of tet(A), tet(B), tet(C), and tet(R) resistance and regulatory genes (Boerlin et al., 2005). Genes for sulfamethoxazole and trimethoprim resistance were also detected in E. coli and included members of sulI/sul1, and sulII and dfrA1, and dhf families, respectively (Singh et al., 2005; Solberg et al., 2006; White et al., 2002). Also identified were integron, transposon, plasmid genes (e.g., intI1, tnpA, and repA), efflux genes, and sanitizer and metal resistance genes predominantly members of the qac, and mer families of efflux systems. Other studies of E. coli isolated from pork, swine, or swine production facilities found similar results (Bass et al., 1999; Walsh and Fanning, 2008). In cases where these studies investigated the genetic structure and specific mechanisms of resistance most were found to be associated with MDR plasmids, class I integrons, or a class I integron located on a plasmid (Bass et al., 1999; Sandvang and Aarestrup, 2000; White et al., 2002; Ajiboye et al., 2009; Fricke et al., 2009b). At least one plasmid replicon was also detected in 41 of the E. coli isolates. These were most often IncF plasmids, including FIB and F, and IncB/O was also detected in many isolates.

The Salmonella isolates in this study had resistance- and mobilization-associated genes detected similar to those found in E. coli. Some specific differences between Salmonella and E. coli were hybridizations of Salmonella DNA to a larger number and wider variety of β-lactam resistance genes. These included probes for bla _CMY, bla _SME, bla _PSE, and bla _SHV homologs. This is consistent with the greater number of β-lactam-resistant Salmonella isolates in this study as well as resistance to a wider variety of β-lactam antimicrobials in the isolates. These results are also comparable to other studies (Villa et al., 2000; Gebreyes and Altier, 2002; Gebreyes and Thakur, 2005; Frye and Fedorka-Cray, 2007; Heider et al., 2009). In previous studies when large numbers of resistances and resistance to extended-spectrum cephalosporins such as ceftiofur were detected, the resistant elements were often found to be encoded on IncA/C plasmids containing the bla _CMY-2 gene, resulting in what has been termed the MDR-AmpC phenotype (Arlet et al., 2006; Frye and Fedorka-Cray, 2007; Zaidi et al., 2007; Zhao et al., 2007). The IncA/C replicon was detected in five Salmonella isolates, two of which also had the bla _CMY gene detected. However, bla _CMY was also detected in three other Salmonella that were not IncA/C replicon positive. These results and comparisons to our previous work suggest that the two bla _CMY gene and IncA/C replicon-positive, ceftiofur-resistant isolates may harbor an MDR-AmpC IncA/C or related plasmid (Frye et al., 2006, 2009; Lindsey et al., 2009, 2010). The other three bla _CMY-positive Salmonella had no plasmid replicons detected, and the gene may be located on an undetected plasmid or elsewhere in the genome. Some Salmonella isolates had other plasmid replicons detected, including FIA, FIB, FIIA, F, HI1, and P, several of which have been reported to carry AR (Carattoli et al., 2001, 2005, 2006; Debroy et al., 2010). In other swine studies, resistances were found to be located in Salmonella Genomic Island 1 (SGI1), which includes a class I integron encoding penta-resistance (Boyd et al., 2001; Doublet et al., 2003, 2004; Ebner et al., 2004; Fluit, 2005). Some Salmonella in the current study hybridized to gene probes found in many SGI1 variants, including aadA2, qacE, floR, tet(A) (which will also hybridize with the tet(G) gene of SGI1), bla _PSE-1, sul1, intI1, and dfrA1 (Boyd et al., 2001; Doublet et al., 2003, 2004; Ebner et al., 2004; Fluit, 2005). Based on these results, many isolates in this study may harbor SGI1.

Many of the resistance genes were found to be shared among isolates from the same sample, but mostly among the closely related E. coli and Salmonella. Not only did these bacteria share many of the same resistance genes, but in almost half of the samples the genes were also shared at a significant level. These data confirm many other studies that have seen similar correlations; however, the large amount of information yielded by the current study is intriguing and may be evidence for a common source of resistances in E. coli and Salmonella, or for transfer of these MDR elements between these species. Genetic elements such as plasmids that could facilitate transferring AR between these bacteria were assayed for, but only detected in a few isolates. Common plasmids detected in both bacteria were F in four samples and FIB in one sample. Although these plasmids have been found to carry resistances, they are not as prevalent as others like IncA/C (Carattoli et al., 2001, 2005, 2006; Debroy et al., 2010). Many other plasmids were detected in both E. coli and Salmonella isolates, but not from the same sample. These data suggest that if exchange of the these genes occurred, in most cases the elements responsible for the transfer were not detected or were already lost in either the donor, the recipient, or both.

A recent study using similar methods to investigate dairy cattle isolates concluded that while common AR genes were detected in E. coli and Salmonella isolates, it was unlikely that multiple large groups of genes were exchanged between these bacteria. This was based upon hierarchical clustering of their AR microarray data that resulted in E. coli and Salmonella isolates forming separate clusters (Scaria et al., 2010). This is similar to the cluster analysis of our data where we also found that isolates grouped into clusters based upon species rather than sample. However, some probes on our array are relatively species specific; thus, even when many common resistance genes are detected in different species, the isolates will still be clustered by the species-specific genes detected because these will be the discriminating factors used by the cluster analysis to group the isolates. Additionally, in our study the microarray also detected common AR mobilization-associated elements such as class I integrons, transposons, and IS elements, which were not assayed by the microarray used in the dairy cattle study (Scaria et al., 2010). Although these elements cannot transfer between bacteria, they are often transferred by conjugal elements to other bacteria where they can integrate into their new host's DNA (Nemergut et al., 2008). Detection of these genes in conjunction with AR genes suggests that integrons in our isolates could have been mobilized by a transfer element between E. coli and Salmonella, followed by the loss of the transfer element. However, it is necessary to avoid over interpretation of the significance at which common genes were detected in E. coli and Salmonella. For example, many of the isolates from different samples also share genes that implicate a common source for resistance genes in the swine environment, or historical exchange of MDR elements rather than recent events or exchange within the animals from which the fecal droppings were collected.

Resistance genes detected in Campylobacter and Enterococcus were also similar to what was found in previous swine studies. The most prevalent resistance genes found in C. coli isolates were tet(O), aphA-3, and cam-1 encoding tetracycline resistance, aminoglycoside resistance, and an endogenous Camplyobacter β-lactamase, respectively (Aarestrup et al., 2000; Jackson et al., 2004b; Thakur and Gebreyes, 2005). The Enterococcus spp. isolates had a far greater number of resistance genes detected, including aad, aac, aphA-3, tet(M) and tet(O) homologs, ermB, vanC, and transposon-related mobilization elements, tnpA, insA, and trans-1, many of which have been previously described (Jackson et al., 2004b). Enterococcus and Campylobacter isolates both hybridized to probes for tet(O). However, in most cases Enterococcus isolates hybridized to both tet(O) and tet(M) probes, whereas C. coli isolates hybridized almost exclusively to the tet(O) probe (Aarestrup et al., 2000; Gibreel et al., 2004; Thakur and Gebreyes, 2005). This is evidence for sharing homologous but not identical tetracycline resistance genes in these bacteria. Moreover, comparison of the C. coli tet(O) sequence in GenBank to the Enterococcus spp. tet(M) sequence found 79% homology by BLAST analysis.

The effects of selective pressure on development, maintenance, and transfer of resistance and MDR elements are well documented in the literature. One of the observations in this study was that sanitizing agent and metal resistance genes were detected in E. coli and Salmonella isolates. These resistance genes are often found to be genetically linked to AR genes by residing on the same genetic element. It is possible that the utilization of sanitizers and metal containing compounds as well as other practices or natural events in the animal environment could be selecting for AR phenotypes in the absence of selection by antibiotics (Fricke et al., 2009a, 2009b; Lindsey et al., 2009; Welch et al., 2009). This could have profound effects on AR bacteria where production practices have begun to rely on metal compounds in feed as an alternative to antimicrobials or in swine facilities and processing plants where sanitizers like quaternary ammonium compounds are used to reduce bacterial contamination in pens, on processing surfaces, or on meat products (Hasman and Aarestrup, 2002; Aarestrup and Hasman, 2004; Pettigrew, 2006). If genetic resistance elements to these compounds are linked to AR genes, withdrawal of antimicrobials may not reduce AR in the bacteria of concern.

This study found that some common resistance genes were shared by isolates from the same swine fecal sample. This was especially true for E. coli and Salmonella, and more rarely for C. coli and Enterococcus spp. possibly indicating that exchange occurs more often between closely related bacteria. A few potential vehicles for resistance gene transfer in E. coli and Salmonella were detected as well as genetic elements associated with mobilization of AR, such as integrons and transposons. Further studies will be required to confirm this exchange, identify the transfer elements, and determine whether AR gene transfer took place recently in the swine environment. Additional analysis of MDR in Salmonella and E. coli animal isolates will allow us to investigate the development, evolution, and spread of MDR genetic elements in animal environments, processing facilities, and food products. This information will improve our understanding of the mechanisms driving AR and enable the development of strategies to reduce AR and promote human and animal health.

Footnotes

Acknowledgments

The authors would like to thank all CAHFSE partners, especially David A. Dargatz, Charles A. Haley, and Christine A. Kopral with the APHIS, USDA, without whom samples could not have been collected. The authors thank Georgina Hidalgo, Alice Wilcher, Mike Asher, Russ Turpin, Jonathan Cudnik, Jovita Haro, Jodie Plumblee, Sandra House, Lari Hiott, and Takiyah Ball for technical assistance.

Disclosure Statement

No competing financial interests exist.

Disclaimer

Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

References

Aarestrup

. Association between the consumption of antimicrobial agents in animal husbandry and the occurrence of resistant bacteria among food animals. Int J Antimicrob Agents, 1999; 12:279–285.

Aarestrup

, Agerso

, Gerner-Smidt

, Madsen

, Jensen

. Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers, and pigs in Denmark. Diagn Microbiol Infect Dis, 2000; 37:127–137.

Aarestrup

, Hasman

. Susceptibility of different bacterial species isolated from food animals to copper sulphate, zinc chloride and antimicrobial substances used for disinfection. Vet Microbiol, 2004; 100:83–89.

Aarestrup

, Oliver

, Burch

. Antimicrobial resistance in swine production. Anim Health Res Rev, 2008a. 9:135–148.

Aarestrup

, Wegener

. The effects of antibiotic usage in food animals on the development of antimicrobial resistance of importance for humans in Campylobacter and Escherichia coli. Microbes Infect, 1999; 1:639–644.

Aarestrup

, Wegener

, Collignon

. Resistance in bacteria of the food chain: epidemiology and control strategies. Expert Rev Anti Infect Ther, 2008b. 6:733–750.

Ajiboye

, Solberg

, Lee

, Raphael

, Debroy

, Riley

. Global spread of mobile antimicrobial drug resistance determinants in human and animal Escherichia coli and Salmonella strains causing community-acquired infections. Clin Infect Dis, 2009; 49:365–371.

Akwar

, Poppe

, Wilson

, Reid-Smith

, Dyck

, Waddington

, Shang

, Dassie

, McEwen

. Risk factors for antimicrobial resistance among fecal Escherichia coli from residents on forty-three swine farms. Microb Drug Resist, 2007; 13:69–76.

Arlet

, Barrett

, Butaye

, Cloeckaert

, Mulvey

, White

. Salmonella resistant to extended-spectrum cephalosporins: prevalence and epidemiology. Microbes Infect, 2006; 8:1945–1954.

10.

Bass

, Liebert

, Lee

, Summers

, White

, Thayer

, Maurer

. Incidence and characterization of integrons, genetic elements mediating multiple-drug resistance, in avian Escherichia coli. Antimicrob Agents Chemother, 1999; 43:2925–2929.

11.

Batchelor

, Hopkins

, Liebana

, Slickers

, Ehricht

, Mafura

, Aarestrup

, Mevius

, Clifton-Hadley

, Woodward

, Davies

, Threlfall

, Anjum

. Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria. Int J Antimicrob Agents, 2008; 31:440–451.

12.

Boerlin

, Reid-Smith

. Antimicrobial resistance: its emergence and transmission. Anim Health Res Rev, 2008; 9:115–126.

13.

Boerlin

, Travis

, Gyles

, Reid-Smith

, Janecko

, Lim

, Nicholson

, McEwen

, Friendship

, Archambault

. Antimicrobial resistance and virulence genes of Escherichia coli isolates from swine in Ontario. Appl Environ Microbiol, 2005; 71:6753–6761.

14.

Boyd

, Peters

, Cloeckaert

, Boumedine

, Chaslus-Dancla

, Imberechts

, Mulvey

. Complete nucleotide sequence of a 43-kilobase genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104 and its identification in phage type DT120 and serovar Agona. J Bacteriol, 2001; 183:5725–5732.

15.

Bruant

, Maynard

, Bekal

, Gaucher

, Masson

, Brousseau

, Harel

. Development and validation of an oligonucleotide microarray for detection of multiple virulence and antimicrobial resistance genes in Escherichia coli. Appl Environ Microbiol, 2006; 72:3780–3784.

16.

Bywater

, Deluyker

, Deroover

, de

, Marion

, McConville

, Rowan

, Shryock

, Shuster

, Thomas

, Valle

, Walters

. A European survey of antimicrobial susceptibility among zoonotic and commensal bacteria isolated from food-producing animals. J Antimicrob Chemother, 2004; 54:744–754.

17.

Carattoli

, Bertini

, Villa

, Falbo

, Hopkins

, Threlfall

. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods, 2005; 63:219–228.

18.

Carattoli

, Miriagou

, Bertini

, Loli

, Colinon

, Villa

, Whichard

, Rossolini

. Replicon typing of plasmids encoding resistance to newer beta-lactams. Emerg Infect Dis, 2006; 12:1145–1148.

19.

Carattoli

, Villa

, Pezzella

, Bordi

, Visca

. Expanding drug resistance through integron acquisition by IncFI plasmids of Salmonella enterica Typhimurium. Emerg Infect Dis, 2001; 7:444–447.

20.

Chen

, Zhao

, McDermott

, Schroeder

, White

, Meng

. A DNA microarray for identification of virulence and antimicrobial resistance genes in Salmonella serovars and Escherichia coli. Mol Cell Probes, 2005; 19:195–201.

21.

Debroy

, Sidhu

, Sarker

, Jayarao

, Stell

, Bell

, Johnson

. Complete sequence of pEC14_114, a highly conserved IncFIB/FIIA plasmid associated with uropathogenic Escherichia coli cystitis strains. Plasmid, 2010; 63:53–60.

22.

de Hoon

, Imoto

, Nolan

, Miyano

. Open source clustering software. Bioinformatics, 2004; 20:1453–1454.

23.

Doublet

, Lailler

, Meunier

, Brisabois

, Boyd

, Mulvey

, Chaslus-Dancla

, Cloeckaert

. Variant Salmonella genomic island 1 antibiotic resistance gene cluster in Salmonella enterica serovar Albany. Emerg Infect Dis, 2003; 9:585–591.

24.

Doublet

, Weill

, Fabre

, Chaslus-Dancla

, Cloeckaert

. Variant Salmonella genomic island 1 antibiotic resistance gene cluster containing a novel 3'-N-aminoglycoside acetyltransferase gene cassette, aac(3)-Id, in Salmonella enterica serovar newport. Antimicrob Agents Chemother, 2004; 48:3806–3812.

25.

Ebner

, Garner

, Mathew

. Class 1 integrons in various Salmonella enterica serovars isolated from animals and identification of genomic island SGI1 in Salmonella enterica var. Meleagridis. J Antimicrob Chemother, 2004; 53:1004–1009.

26.

Eisen

, Spellman

, Brown

, Botstein

. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA, 1998; 95:14863–14868.

27.

Fluit

. Towards more virulent and antibiotic-resistant Salmonella? FEMS Immunol Med Microbiol, 2005; 43:1–11.

28.

Fricke

, McDermott

, Mammel

, Zhao

, Johnson

, Rasko

, Fedorka-Cray

, Pedroso

, Whichard

, LeClerc

, White

, Cebula

, Ravel

. Antimicrobial resistance-conferring plasmids with similarity to virulence plasmids from avian pathogenic Escherichia coli strains in Salmonella enterica serovar Kentucky isolates from poultry. Appl Environ Microbiol, 2009a. 75:5963–5971.

29.

Fricke

, Welch

, McDermott

, Mammel

, LeClerc

, White

, Cebula

, Ravel

. Comparative genomics of the IncA/C multidrug resistance plasmid family. J Bacteriol, 2009b. 191:4750–4757.

30.

Frye

, Fedorka-Cray

. Prevalence, distribution and characterisation of ceftiofur resistance in Salmonella enterica isolated from animals in the USA from 1999 to 2003. Int J Antimicrob Agents, 2007; 30:134–142.

31.

Frye

, Jesse

, Long

, Rondeau

, Porwollik

, McClelland

, Jackson

, Englen

, Fedorka-Cray

. DNA microarray detection of antimicrobial resistance genes in diverse bacteria. Int J Antimicrob Agents, 2006; 27:138–151.

32.

Frye

, Lindsey

, Rondeau

, Porwollik

, Long

, McClelland

, Jackson

, Englen

, Meinersmann

, Berrang

, Davis

, Barrett

, Turpin

, Thitaram

, Fedorka-Cray

. Development of a DNA microarray to detect antimicrobial resistance genes identified in the National Center for Biotechnology Information Database. Microb Drug Resist, 2009; 16:9–19.

33.

Gebreyes

, Altier

. Molecular characterization of multidrug-resistant Salmonella enterica subsp. enterica serovar Typhimurium isolates from swine. J Clin Microbiol, 2002; 40:2813–2822.

34.

Gebreyes

, Davies

, Morrow

, Funk

, Altier

. Antimicrobial resistance of Salmonella isolates from swine. J Clin Microbiol, 2000; 38:4633–4636.

35.

Gebreyes

, Davies

, Turkson

, Morrow

, Funk

, Altier

, Thakur

. Characterization of antimicrobial-resistant phenotypes and genotypes among Salmonella enterica recovered from pigs on farms, from transport trucks, and from pigs after slaughter. J Food Prot, 2004; 67:698–705.

36.

Gebreyes

, Thakur

. Multidrug-resistant Salmonella enterica serovar Muenchen from pigs and humans and potential interserovar transfer of antimicrobial resistance. Antimicrob Agents Chemother, 2005; 49:503–511.

37.

Gebreyes

, Thakur

, Morrow

. Campylobacter coli: prevalence and antimicrobial resistance in antimicrobial-free (ABF) swine production systems. J Antimicrob Chemother, 2005; 56:765–768.

38.

Gibreel

, Tracz

, Nonaka

, Ngo

, Connell

, Taylor

. Incidence of antibiotic resistance in Campylobacter jejuni isolated in Alberta, Canada, from 1999 to 2002, with special reference to tet(O)-mediated tetracycline resistance. Antimicrob Agents Chemother, 2004; 48:3442–3450.

39.

Hasman

, Aarestrup

. tcrB, a gene conferring transferable copper resistance in Enterococcus faecium: occurrence, transferability, and linkage to macrolide and glycopeptide resistance. Antimicrob Agents Chemother, 2002; 46:1410–1416.

40.

Heider

, Hoet

, Wittum

, Khaitsa

, Love

, Huston

, Morley

, Funk

, Gebreyes

. Genetic and phenotypic characterization of the bla(CMY) gene from Escherichia coli and Salmonella enterica isolated from food-producing animals, humans, the environment, and retail meat. Foodborne Pathog Dis, 2009; 6:1235–1240.

41.

Jackson

, Fedorka-Cray

, Barrett

. Use of a genus- and species-specific multiplex PCR for identification of enterococci. J Clin Microbiol, 2004a. 42:3558–3565.

42.

Jackson

, Fedorka-Cray

, Barrett

, Ladely

. Effects of tylosin use on erythromycin resistance in enterococci isolated from swine. Appl Environ Microbiol, 2004b. 70:4205–4210.

43.

Jackson

, Fedorka-Cray

, Barrett

, Ladely

. High-level aminoglycoside resistant enterococci isolated from swine. Epidemiol Infect, 2005; 133:367–371.

44.

Jakobsen

, Kurbasic

, Skjot-Rasmussen

, Ejrnaes

, Porsbo

, Pedersen

, Jensen

, Emborg

, Agerso

, Olsen

, Aarestrup

, Frimodt-Moller

, Hammerum

. Escherichia coli isolates from broiler chicken meat, broiler chickens, pork, and pigs share phylogroups and antimicrobial resistance with community-dwelling humans and patients with urinary tract infection. Foodborne Pathog Dis, 2010; 7:537–547.

45.

Kozak

, Pearl

, Parkman

, Reid-Smith

, Deckert

, Boerlin

. Distribution of sulfonamide resistance genes in Escherichia coli and Salmonella isolates from swine and chickens at abattoirs in Ontario and Quebec, Canada. Appl Environ Microbiol, 2009; 75:5999–6001.

46.

Lindsey

, Fedorka-Cray

, Frye

, Meinersmann

. IncA/C plasmids are prevalent in multidrug-resistant Salmonella enterica isolates. Appl Environ Microbiol, 2009; 75:1908–1915.

47.

Lindsey

, Frye

, Fedorka-Cray

, Welch

, Meinersmann

. An oligonucleotide microarray to characterize multidrug resistant plasmids. J Microbiol Methods, 2010; 81:96–100.

48.

Liu

, Han

, Huang

, Zhu

. Development and evaluation of 16S rDNA microarray for detecting bacterial pathogens in cerebrospinal fluid. Exp Biol Med (Maywood), 2005; 230:587–591.

49.

Luo

, Pereira

, Sahin

, Lin

, Huang

, Michel

, Zhang

. Enhanced in vivo fitness of fluoroquinolone-resistant Campylobacter jejuni in the absence of antibiotic selection pressure. Proc Natl Acad Sci USA, 2005; 102:541–546.

50.

, Wang

, Yu

, Zhang

, Liu

. Detection of antimicrobial resistance genes of pathogenic Salmonella from swine with DNA microarray. J Vet Diagn Invest, 2007; 19:161–167.

51.

McDermott

, Zhao

, Wagner

, Simjee

, Walker

, White

. The food safety perspective of antibiotic resistance. Anim Biotechnol, 2002; 13:71–84.

52.

Michael

, Gillings

, Holmes

, Hughes

, Andrew

, Holley

, Stokes

. Mobile gene cassettes: a fundamental resource for bacterial evolution. Am Nat, 2004; 164:1–12.

53.

NCCLS. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals. Wayne, PA, 2002.

54.

NCCLS. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Wayne, PA, 2003.

55.

Nemergut

, Robeson

, Kysela

, Martin

, Schmidt

, Knight

. Insights and inferences about integron evolution from genomic data. BMC Genomics, 2008; 9:261.

56.

Perreten

, Vorlet-Fawer

, Slickers

, Ehricht

, Kuhnert

, Frey

. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J Clin Microbiol, 2005; 43:2291–2302.

57.

Pettigrew

. Reduced use of antibiotic growth promoters in diets fed to weanling pigs: dietary tools, part 1. Anim Biotechnol, 2006; 17:207–215.

58.

Phillips

. Withdrawal of growth-promoting antibiotics in Europe and its effects in relation to human health. Int J Antimicrob Agents, 2007; 30:101–107.

59.

Phillips

, Casewell

, Cox

, De Groot

, Friis

, Jones

, Nightingale

, Preston

, Waddell

. Antibiotic use in animals. J Antimicrob Chemother, 2004; 53:885.

60.

Porwollik

, Frye

, Florea

, Blackmer

, McClelland

. A non-redundant microarray of genes for two related bacteria. Nucleic Acids Res, 2003; 31:1869–1876.

61.

Porwollik

, Wong

, Sims

, Schaaper

, DeMarini

, McClelland

. The DeltauvrB mutations in the Ames strains of Salmonella span 15 to 119 genes. Mutat Res, 2001; 483:1–11.

62.

Rosengren

, Waldner

, Reid-Smith

. Associations between antimicrobial resistance phenotypes, antimicrobial resistance genes, and virulence genes of fecal Escherichia coli isolates from healthy grow-finish pigs. Appl Environ Microbiol, 2009; 75:1373–1380.

63.

Saldanha

. Java Treeview—extensible visualization of microarray data. Bioinformatics, 2004; 20:3246–3248.

64.

Sandvang

, Aarestrup

. Characterization of aminoglycoside resistance genes and class 1 integrons in porcine and bovine gentamicin-resistant Escherichia coli. Microb Drug Resist, 2000; 6:19–27.

65.

Scaria

, Warnick

, Kaneene

, May

, Teng

, Chang

. Comparison of phenotypic and genotypic antimicrobial profiles in Escherichia coli and Salmonella enterica from the same dairy cattle farms. Mol Cell Probes, 2010; 24:325–345.

66.

Singh

, Schroeder

, Meng

, White

, McDermott

, Wagner

, Yang

, Simjee

, Debroy

, Walker

, Zhao

. Identification of antimicrobial resistance and class 1 integrons in Shiga toxin-producing Escherichia coli recovered from humans and food animals. J Antimicrob Chemother, 2005; 56:216–219.

67.

Solberg

, Ajiboye

, Riley

. Origin of class 1 and 2 integrons and gene cassettes in a population-based sample of uropathogenic Escherichia coli. J Clin Microbiol, 2006; 44:1347–1351.

68.

Stern

, Wojton

, Kwaitek

. A differential-selective medium and dry ice-generated atmosphere for recovery of Campylobacter jejuni. J Food Prot, 1992; 55:514–517.

69.

Thakur

, Gebreyes

. Campylobacter coli in swine production: antimicrobial resistance mechanisms and molecular epidemiology. J Clin Microbiol, 2005; 43:5705–5714.

70.

Thakur

, Tadesse

, Morrow

, Gebreyes

. Occurrence of multidrug resistant Salmonella in antimicrobial-free (ABF) swine production systems. Vet Microbiol, 2007; 125:362–367.

71.

van den Bogaard

, Stobberingh

. Epidemiology of resistance to antibiotics. Links between animals and humans. Int J Antimicrob Agents, 2000; 14:327–335.

72.

Varga

, Rajic

, McFall

, Avery

, Reid-Smith

, Deckert

, Checkley

, McEwen

. Antimicrobial resistance in generic Escherichia coli isolated from swine fecal samples in 90 Alberta finishing farms. Can J Vet Res, 2008; 72:175–180.

73.

Villa

, Pezzella

, Tosini

, Visca

, Petrucca

, Carattoli

. Multiple-antibiotic resistance mediated by structurally related IncL/M plasmids carrying an extended-spectrum beta-lactamase gene and a class 1 integron. Antimicrob Agents Chemother, 2000; 44:2911–2914.

74.

Walsh

, Fanning

. Antimicrobial resistance in foodborne pathogens—a cause for concern? Curr Drug Targets, 2008; 9:808–815.

75.

Wassenaar

, Silley

. Antimicrobial resistance in zoonotic bacteria: lessons learned from host-specific pathogens. Anim Health Res Rev, 2008; 9:177–186.

76.

Wegener

, Aarestrup

, Gerner-Smidt

, Bager

. Transfer of antibiotic resistant bacteria from animals to man. Acta Vet Scand Suppl, 1999; 92:51–57.

77.

Welch

, Evenhuis

, White

, McDermott

, Harbottle

, Miller

, Griffin

, Wise

. IncA/C plasmid-mediated florfenicol resistance in the catfish pathogen Edwardsiella ictaluri. Antimicrob Agents Chemother, 2009; 53:845–846.

78.

White

, Zhao

, McDermott

, Ayers

, Gaines

, Friedman

, Wagner

, Meng

, Needle

, Davis

, Debroy

. Characterization of antimicrobial resistance among Escherichia coli O111 isolates of animal and human origin. Microb Drug Resist, 2002; 8:139–146.

79.

Zaidi

, Leon

, Canche

, Perez

, Zhao

, Hubert

, Abbott

, Blickenstaff

, McDermott

. Rapid and widespread dissemination of multidrug-resistant blaCMY-2 Salmonella Typhimurium in Mexico. J Antimicrob Chemother, 2007; 60:398–401.

80.

Zhang

, Sahin

, McDermott

, Payot

. Fitness of antimicrobial-resistant Campylobacter and Salmonella. Microbes Infect, 2006; 8:1972–1978.

81.

Zhao

, McDermott

, White

, Qaiyumi

, Friedman

, Abbott

, Glenn

, Ayers

, Post

, Fales

, Wilson

, Reggiardo

, Walker

. Characterization of multidrug resistant Salmonella recovered from diseased animals. Vet Microbiol, 2007; 123:122–132.

82.

Zou

, Frye

, Chang

, Liu

, Cerniglia

, Nayak

. Microarray analysis of antimicrobial resistance genes in Salmonella enterica from preharvest poultry environment. J Appl Microbiol, 2009; 107:906–914.

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