Prevalence of Enterohemorrhagic Escherichia coli O26,O45,O103,O111,O121,O145,and O157 on Hides and Preintervention Carcass Surfaces of Feedlot Cattle at Harvest

Abstract

Cattle hides are a main source of enterohemorrhagic Escherichia coli (EHEC) contamination of beef carcasses. The objectives of this study were to (1) determine the prevalence of “top 6” non-O157 plus O157:H7 EHEC (EHEC-7) on feedlot cattle hides and their matched preintervention carcasses; (2) assess the agreement among detection methods for these matrices; and (3) conduct a molecular risk assessment of EHEC-7 isolates. Samples from 576 feedlot cattle were obtained at a commercial harvest facility and tested for EHEC-7 by a culture-based method and the polymerase chain reaction/mass spectrometry–based NeoSEEK^™ STEC Detection and Identification test (NS). Prevalence data were analyzed with generalized linear mixed models. The cumulative prevalence of EHEC-7 in hide samples as detected by NS was 80.7%, with a distribution of 49.9%, O145; 37.1%, O45; 12.5%, O103; 11.0%, O157; 2.2%, O111; 2.0%, O121; and 0.2%, O26. In contrast, the cumulative prevalence of EHEC-7 in hide samples by culture was 1.2%, with a distribution of 0.6%, O157; 0.4%, O26; 0.2%, O145; and 0%, O45, O103, O111, and O121. The cumulative prevalence of EHEC-7 on matched preintervention carcasses as detected by NS was 6.0%, with a distribution of 2.8%, O157; 1.6%, O145; 1.2%, O103; 1.1%, O45; 0.2%, O26; and 0.0%, O111 and O121. Although the culture-based method detected fewer positive hide samples than NS, it detected EHEC in five hide samples that tested negative for the respective organism by NS. McNemar's chi-square tests indicated significant (p<0.05) disagreement between methods. All EHEC-7 isolates recovered from hides were seropathotype A or B, with compatible virulence gene content. This study indicates that “top 6” and O157:H7 EHEC are present on hides, and to a lesser extent, preintervention carcasses of feedlot cattle at harvest. However, continued improvement in non-O157 detection methods is needed for accurate estimation of prevalence, given the discordant results across protocols.

Introduction

Shiga toxin (Stx)–producing Escherichia coli (STEC) are major pathogens of humans and most commonly acquired through the consumption of contaminated food or water (Cole et al., 2014; Luna-Gierke et al., 2014). E. coli O157:H7 is the prototype of STEC further classified as enterohemorrhagic E. coli (EHEC), which by definition contain genes for Stx (stx) and products encoded on the locus of enterocyte effacement pathogenicity island, most notably intimin (eae) (Kaper et al., 2004). All STEC not of the O157 O-antigen type are commonly called non-O157 (Bettelheim, 2007). From 1983 to 2002, 6 serogroups (viz., O26, O45, O103, O111, O121, and O145) accounted for 71% of the reported non-O157 STEC cases in the United States (Brooks et al., 2005); STEC belonging to these serogroups have become known as the “top 6” (Hegde et al., 2012; Conrad et al., 2014). A total of 84% of the “top 6” isolates in the cases reported by Brooks et al. (2005) were EHEC.

Collectively, the “top 6” non-O157 plus O157:H7 (hereafter referred to as EHEC-7) were responsible for 92.3% of the human cases of STEC infection in the United States from 2000 to 2010 (Gould et al., 2013). E. coli O157:H7 was declared an adulterant in raw ground beef in 1994 and in raw, nonintact beef in 1999 by the United States Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS). The “top 6” non-O157 STEC were declared adulterants in raw, nonintact beef by the USDA-FSIS in 2011. Based on the requirement for stx and eae detection in a “top 6” isolate for qualification as an adulterant (USDA FSIS, 2014), these organisms are also by definition EHEC.

STEC mainly contaminate beef through transfer from fecally contaminated hides to carcass surfaces during slaughter (Elder et al., 2000; Arthur et al., 2002; Keen and Elder, 2002; Barkocy-Gallagher et al., 2003; Arthur et al., 2004; Koohmaraie et al., 2005; Schmidt et al., 2012). The prevalence of O157 and non-O157 STEC on de-hided carcass surfaces has been reported, but little information is available on the “top 6.” The objectives of this study were to (1) determine the prevalence of EHEC-7 on matched hides and de-hided preintervention carcass surface samples obtained from commercial feedlot cattle at harvest; (2) compare the performance capability and agreement of a culture-based method with that of a polymerase chain reaction (PCR)/mass spectrometry–based method for detection of EHEC-7 in enriched hide and carcass sponge samples, and a PCR/mass spectrometry–based method to a commercial immunomagnetic/PCR–based method for enriched carcass sponge samples; and (3) conduct a molecular risk assessment (MRA) of EHEC-7 isolates obtained from hides.

Materials and Methods

Study design and sample collection

Cattle from a commercial beef feedlot in the central United States were followed through harvest using a repeated cross-sectional study design. Matched samples from the hides and carcass surfaces of 576 cattle (24 pens, 24 cattle/pen, 2 pens/wk for 12 wks, from June to August 2013) were obtained at a processing plant using a modified protocol for E. coli O157:H7 sampling (USDA FSIS, 2005). While in lairage, cattle were sprayed with a commercial bacteriophage solution (Finalyse^®; Elanco Animal Health, Greenfield, IN) that is lytic for O157:H7 (Sillankorva et al., 2012). Stunned animals were sprayed with a high-pressure ambient water rinse, shackled, and placed onto the overhead rail for exsanguination. Afterward, the hides were sampled immediately before the carcasses entered the first hide-on wash cabinet.

Hide samples were collected using 11.5×23.0-cm sponges (Speci-Sponge^®; Nasco, Fort Atkinson, WI) premoistened with 35 mL of buffered peptone water (BPW). Sponges were used to sample an area of 1000 cm², 15 cm from the midline at the level of the diaphragm.

From each de-hided carcass, two surface samples were collected before the first carcass cabinet wash occurred. The first covered a 1000-cm² area in the brisket–short plate region using a Speci-Sponge^® with 15 mL of BPW, following the procedure used for hides. The second was from a 3000-cm² area in the lateral hock and round-rump regions. These 2 sponges and their buffer volumes (representing a combined 4000 cm² area per carcass) were combined into a single Whirl-Pak^® bag. Samples were shipped overnight on ice to the laboratories.

Culture of hide and carcass samples

Hide and carcass sponge samples were processed and analyzed within 24 h after collection. Ninety milliliters of E. coli broth (EC; Oxoid Ltd., Hampshire, UK) was added to each hide sponge sample, and these were incubated at 40°C for 6 h. After incubation, separate broth aliquots were subjected to immunomagnetic separation (IMS) using a KingFisher^™ Flex Magnetic Particle Processor (Thermo Scientific, Waltham, MA) with anti-O157 Dynabeads^® (Invitrogen, Carlsbad, CA), and IMS beads for E. coli O26, O45, O103, O111, O121, and O145 (Abraxis LLC, Warminster, PA). Washed IMS beads were spread onto Possé differential agar (Possé et al., 2008) modified (mPossé) by reducing the concentration of novobiocin and potassium tellurite to 5.0 and 0.5 mg/L, respectively. mPossé plates were each spread-inoculated with 1 IMS treatment and incubated for 18 h at 37°C. Six or fewer colonies per mPossé plate were picked; on plates inoculated with O26, O45, O103, O111, or O157 IMS-treated cultures, red- or blue-purple colonies were picked and for O121 or O145 IMS-treated cultures, red- or blue-purple, and green colonies were picked. Colonies were streaked for isolation on 5% sheep blood agar (Remel, Lenexa, KS), and incubated for 15 h at 37°C. Isolated colonies were picked, suspended in 50 μL of ultrapure water, and boiled for 10 min for use as DNA template in PCR assays.

Eighty milliliters of EC broth was added to each carcass sponge sample, and incubated at 42°C for 8 h. Following incubation, a 1-mL aliquot of each enrichment broth sample was tested using the Biocontrol Assurance GDS^™ MPX Top 7 STEC system (GDS) (Biocontrol Systems, Inc., Bellevue, WA) following the manufacturer's protocol. The GDS system consists of a proprietary IMS that targets the EHEC-7 serogroups (i.e., the seven O-group antigens) followed by PCR for detection of eae, stx ₁, and stx ₂, with EHEC-6 and EHEC O157 presumptive positive or negative results generated. An additional 1-mL aliquot of each enrichment broth sample was placed into 1.5-mL centrifuge tubes and stored at −80°C for later shipment to GeneSeek^® Inc. (Lincoln, NE) for NeoSEEK™ analysis. Enrichment broths that tested negative by GDS were not cultured further. Those that tested presumptive positive by GDS were subjected to IMS and plated on mPossé agar as described above for the hide samples, with the exception that IMS was done manually. From each mPossé plate, ≤6 isolated red- or blue-purple, and green colonies were picked and subcultured onto trypticase soy agar (TSA) to ensure isolation. TSA-isolated colonies representing those from each mPossé plate were picked, pooled, and tested by GDS. Any pools testing positive for EHEC-6 or EHEC O157 by GDS were to be tested by 11-plex PCR as described below for the hide samples.

Detection and characterization of EHEC isolates from hide samples

From each hide sample culture, ≤6 colonies from mPossé plates were pooled and tested by multiplex PCR for EHEC-7 serogroups. Isolates in positive pools were tested individually by 11-plex PCR for genes representing each of the EHEC-7 serogroups (wzx, wbq, or rfbE), plus stx ₁, stx ₂, eae, and EHEC-hemolysin (ehxA) using methods previously described (Bai et al., 2010; Paddock et al., 2012). H-antigen typing (fliC) was conducted using the methods of Wang et al. (2003). Serogroups for EHEC-7-positive isolates were confirmed by latex agglutination tests (Abraxis LLC, Warminster, PA). Isolates were further characterized by MRA PCR for nleA, nleB, nleB2, nleC, nleD, nleE, nleF, nleG, nleG2-1, nleG2-3, nleG5-2, nleG6-2, nleG9, nleH1-1, nleH1-2, and ent genes, using primers and conditions as previously described (Coombes et al., 2008). In addition, isolates were tested for eae subtypes and putative virulence factor genes, viz., Z4321 (Salmonella enterica serovar Typhimurium [pagC]), Z4326 (Shigella flexneri enterotoxin 2 [sen]), and Z4332 and Z4333 (EHEC factor for adherence [efa1]) as previously described (Karmali et al., 2003; Blanco et al., 2004). In the respective PCR assays, water was used as a negative control. E. coli strain 933 (O157:H7) was used as a positive control for nle, pagC, sen, efa1, and fliC. For the 11-plex PCR, the following strains were used for the respective O group: DEC10B (O26:H11); B8227-C8 (O45:H2); 15612 (O103:H11); 7726 (O111:NT); 4190 (O121:NT); 1234 (O145:H28); and 86-24 (O157:H7).

Detection of EHEC by the NeoSEEK^™ STEC Detection and Identification (NS) test

Enriched hide and carcass sponge sample aliquots stored at −80°C were shipped overnight or directly delivered for EHEC-7 testing by the NS (Neogen^® Corp., Lansing, MI) test, a PCR/mass spectrometry–based analytical method at GeneSeek^® Inc. NS results were reported by the company as confirmed positive or negative. Based on NS data, a positive result for EHEC was inferred by the concurrent detection of stx, targeted O-group single nucleotide polymorphism, specific eae subtypes, and several other virulence markers using a total of 70 independent markers. Identifying combinations of eae subtypes and EHEC-7 O group markers were as follows: eae-β with O26; eae-ɛ with O45, O103, or O121; eae-γ2 with O111; and eae-γ1 with O145 or O157.

Statistical analysis

Adjusted sample-level prevalence estimates for each EHEC serogroup, using the definitions for a positive test result described above, were obtained from model intercepts for each detection protocol (i.e., culture and NS for hide, and GDS and NS for carcass samples). Generalized linear mixed models were fitted using restricted pseudo-likelihood estimation, Newton-Raphson and Ridging optimization, binary distribution, logit link, and Kenward-Rogers degrees of freedom (PROC GLIMMIX, SAS 9.3, SAS Institute Inc., Cary, NC). Random effects of pens nested within sampling week were used to adjust prevalence estimates for the lack of independence resulting from collecting multiple samples from each study pen across multiple sampling weeks.

The overall agreement on detection of EHEC groups between the culture-based method and NS on hide samples, and between GDS and NS on carcasses, beyond that due to chance, was determined by computing the Cohen's κ coefficient and the McNemar's chi-square test (Dohoo et al., 2009). The degree of agreement was interpreted based on the scale proposed by Landis and Koch (1977). In addition, a test of proportions, based on Z tests, was used to compare the proportion of EHEC-7 positives obtained from hide and carcass surface samples based on NS.

Results

Detection of EHEC in hide sponge samples by culture-based and multiplex PCR assays

Of the 576 hide sponge samples cultured, 476 were tested by multiplex PCR, whereas 100 were not tested due to inadequate DNA concentration. At least 1 targeted O-group positive organism (which included both STEC and non-STEC) was isolated from 248 of the 476 hide samples (52.1%) as tested by 11-plex PCR on individual colonies. In descending order, the proportion of hide samples testing positive for the 7 targeted O-group genes (which included both STEC and non-STEC) by PCR following enrichment broth culture was 138/476 (29.0%), O103; 124/476 (26.1%), O157; 52/476 (10.9%), O26; 18/476 (3.8%), O45; 8/476 (1.7%), O111; and 3/476 (0.6%) each, O121 and O145. The proportion of hide samples in which the targeted virulence genes were identified by PCR in these cultures was 25/476 (5.3%), stx; 19/476 (4.0%), eae; and 51/476 (10.7%), ehxA (Table 1). Eight EHEC-7 isolates were recovered by the culture-based protocol from 6/476 (1.3%) of the samples. These isolates included 2 O26:[H11], 1 O145:[H28], and 5 O157:H7 (Table 2). No EHEC-6 or EHEC O157 isolates were obtained from the carcass samples by the culture-based method.

Table 1.

Detection of Enterohemorrhagic Escherichia coli (EHEC) O26, O45, O103, O111, O121, O145, and O157 in Cattle Hide Samples by a Culture-Based Protocol and NeoSEEK ^™ Shiga Toxin–Producing Escherichia coli Detection and Identification Test (NS)

	No. (%) of samples
Detection method	Total ^a	O-group positive ^b	stx positive ^c	eae positive	ehxA positive	EHEC-7 positive
Culture	476 (100)	248 (52.1)	25 (5.3)	19 (4.0)	51 (10.7)	6 (1.3)
NS	576 (100)	576 (100)	574 (99.7)	576 (100)	NA	451 (78.3)

A total of 100 hide samples had inadequate DNA concentration for 11-plex polymerase chain reaction; hence, the culture-based protocol could only be completed on 476 samples.

Samples that isolated one or more O-group positive colonies from serogroups O26, O45, O103, O111, O121, O145 and O157.

Samples positive for stx ₁ and/or stx ₂.

NA, not applicable.

Table 2.

Molecular Risk Assessment of Enterohemorrhagic Escherichia coli (EHEC) Hide Isolates by Polymerase Chain Reaction Assay

Isolate	Sample no.	Serotype	stx^a	eae^b	No. of detected nle genes ^c	pagC^d	sen^e	efa1^f
3-O157-3	3	O157:[H7]	2	γ1	16	+	+	+
1379D	293	O157:[H7]	2	γ1	16	+	+	+
1568B	320	O157:[H7]	2	γ1	16	+	+	+
1379B	293	O157:[H7]	2	γ1	15	+	+	+
3-8G	320	O157:[H7]	2	γ1	15	+	+	+
1394E	296	O26:[H11]	1	β1	15	–	+	+
400D	154	O26:[H11]	1	β1	12	–	+	+
860B	219	O145:[H28]	2	γ1	7	–	+	+

Shiga toxin type.

Intimin type and subtype.

Nonlocus of enterocyte effacement genes.

Detection of putative virulence gene Z4321, homologue of Salmonella enterica serovar Typhimurium (pagC), originally reported on pathogenicity island OI-122 of EHEC O157:H7 strain EDL 933.

Detection of putative virulence gene Z4326, homologue of Shigella flexneri enterotoxin 2 (sen), originally reported on pathogenicity island OI-122 of EHEC O157:H7 strain EDL 933.

Detection of putative virulence genes Z4332 and Z4333 (EHEC factor for adherence [efa1]), originally reported on pathogenicity island OI-122 of EHEC O157:H7 strain EDL 933.

Characterization of EHEC-7 hide isolates

The results of molecularly serotyping and testing for virulence genes and eae subtype in EHEC-7 isolates from hides are shown in Table 2. All EHEC O157:H7 isolates were positive for 15 or 16 nle genes in the MRA, whereas EHEC non-O157 isolates were positive for 7–15 of these genes.

Detection of EHEC on hides and carcasses by NS

The proportion of hide sponge samples testing positive for EHEC-7 by NS was 451/576 (78.3%, Table 3). In descending order, the proportions of these samples testing positive for the different EHEC were 282/576 (49.0%), O145; 227/576 (39.4%), O45; 107/576 (18.6%), O103; 83/576 (14.4%), O157; 14/576 (2.4%), O121; 13/576 (2.3%), O111; and 3/576 (0.5%), O26. Although the NS test detected a higher proportion of positive samples than the culture-based method, five hide samples from which EHEC isolates were obtained by culture tested negative for the respective serogroup by NS. NS detected EHEC O145 from the remaining hide sample from which an EHEC O145 organism was isolated (Table 2). By interpreting in-parallel the combined results of NS and the culture-based method, 78.5% of the hide samples (452/576) were positive for EHEC-7.

Table 3.

Crude Proportions and Model-Adjusted Cumulative Prevalence Estimates of Enterohemorrhagic Escherichia coli (EHEC) O26, O45, O103, O111, O121, O145, and O157 in Cattle Hide Samples as Detected by Culture-Based Protocol and NeoSEEK ^™ STEC Detection and Identification Test (NS)

	Culture			NS
	Total n=476 ^a			Total n=576
Serogroup	No. samples positive (%)	Mean prevalence, %	95% CI	No. samples positive (%)	Mean prevalence, %	95% CI
EHEC O26	2 (0.42)	0.42	0.11–1.66	3 (0.52)	0.21	0.03–1.48
EHEC O45	0 (0.00)	0.00	0.00–0.00	227 (39.41)	37.09	25.24–50.73
EHEC O103	0 (0.00)	0.00	0.00–0.00	107 (18.58)	12.48	6.87–21.59
EHEC O111	0 (0.00)	0.00	0.00–0.00	13 (2.26)	2.23	1.43–3.45
EHEC O121	0 (0.00)	0.00	0.00–0.00	14 (2.43)	1.98	0.98–3.99
EHEC O145	1 (0.21)	0.21	0.03–1.48	282 (48.96)	49.88	38.39–61.39
EHEC O157	3 (0.63)	0.63	0.20–1.94	83 (14.41)	11.02	6.73–17.52
EHEC-6	3 (0.63)	0.63	0.20–1.93	429 (74.48)	77.53	68.47–84.58
EHEC-7	6 (1.26)	1.24	0.54–2.84	451 (78.30)	80.71	73.27–86.46

A total of 100 hide samples had inadequate DNA concentration for 11-plex polymerase chain reaction; hence, the culture-based protocol could be completed on only 476 samples.

The proportion of preintervention de-hided carcass sponge samples testing positive by NS for EHEC-7 was 36/576 (6.3%, Table 4). In descending order, the proportions of these samples testing positive for the different EHEC serogroups was 16/576 (2.8%), O157; 11/576 (1.9%), O145; 10/576 (1.7%), O103; 8/576 (1.4%), O45; 2/576 (0.3%), O26; 0/576 (0.0%) each, for O111 and O121. Based on GDS, 165/576 (28.6%) and 86/576 (14.9%) of the carcass samples were presumptive positive for the “top 6” EHEC and EHEC O157, respectively. Based on NS, the proportion of EHEC-7 positives in hide sponge samples was significantly higher (p<0.05) than in carcass surface samples.

Table 4.

Crude Proportions and Model-Adjusted Cumulative Prevalence Estimates of Enterohemorrhagic Escherichia coli (EHEC) O26, O45, O103, O111, O121, O145, and O157 in Preintervention Carcass Samples by Biocontrol Assurance GDS ^™ MPX Top 7 STEC System (GDS) and NeoSEEK ^tm STEC Detection and Identification Test (NS)

	GDS			NS
	Total n=576			Total n=576
Serogroup or virulence gene	No. samples positive (%)	Mean prevalence, %	95% CI	No. samples positive (%)	Mean prevalence, %	95% CI
EHEC O26	NA	NA	NA	2 (0.35)	0.21	0.03–1.40
EHEC O45	NA	NA	NA	8 (1.39)	1.13	0.47–2.71
EHEC O103	NA	NA	NA	10 (1.74)	1.23	0.50–2.99
EHEC O111	NA	NA	NA	0.00	0.00	0.00–0.00
EHEC O121	NA	NA	NA	0.00	0.00	0.00–0.00
EHEC O145	NA	NA	NA	11 (1.91)	1.58	0.74–3.36
EHEC O157	86 (14.93)	13.13	9.02–18.73	16 (2.78)	2.78	1.71–4.49
EHEC-6	165 (28.65)	24.83	16.72–35.21	24 (4.17)	3.96	2.46–6.31
EHEC-7	NA	NA	NA	36 (6.25)	5.98	4.02–8.80
stx	NA	NA	NA	349 (60.59)	66.37	43.14–83.70
stx ₁	118 (20.49)	15.69	9.55–24.71	NA	NA	NA
stx ₂	138 (23.96)	21.89	15.91–29.33	NA	NA	NA
eae	385 (66.84)	71.69	58.02–82.27	398 (69.10)	74.52	60.17–84.99

NA, not applicable; STEC, Shiga toxin–producing Escherichia coli.

Model-adjusted cumulative prevalence estimates of EHEC on hides and carcasses

The model-adjusted cumulative prevalence estimates of EHEC-7 in hide sponge samples based on the culture-based method and NS are shown in Table 3. The model-adjusted cumulative prevalence estimate of EHEC-7 in hide sponge samples as detected by NS was 80.7%, with a distribution of 49.9%, O145; 37.1%, O45; 12.5%, O103; 11.0%, O157; 2.2%, O111; 2.0%, O121; and 0.2%, O26. In contrast, the model-adjusted cumulative prevalence estimate of EHEC-7 in hide samples based on culture was 1.2%, with a distribution of 0.6%, O157; 0.4%, O26; 0.2%, O145; and 0% for the remaining serogroups.

The model-adjusted cumulative prevalence estimate of EHEC-6 and EHEC O157 in carcass sponge samples based on GDS was 24.8% and 13.1%, respectively (Table 4). In contrast, the model-adjusted cumulative prevalence estimates of EHEC-6 and EHEC-7 in carcass sponge samples as detected by NS were 4.0% and 6.0%, respectively, with a distribution of 2.8%, O157; 1.6%, O145; 1.2%, O103; 1.1%, O45; 0.2%, O26; and 0.0%, O111 and O121.

Comparison of culture-based method versus NS for EHEC-7 detection in hide samples and GDS versus NS for carcass samples

The McNemar's chi-square tests comparing the culture-based method and NS results on hide samples were statistically significant (p<0.05) for all EHEC groups, indicating a serious disagreement between the tests, except for EHEC O26. The κ coefficient for the comparison of the culture-based method and NS for EHEC O26 (κ=−0.005; 95% CI=−0.0099, −0.0003) indicated a poor agreement between the tests. The McNemar's chi-square tests comparing GDS and NS on carcass samples for detection of EHEC O157 and EHEC-6 were statistically significant (p<0.05), indicating disagreement between the tests.

Discussion

Although the results of this study were derived from only one plant, one feedlot, and one period, they suggest that EHEC-7, as detected by NS, are ubiquitous on hides of commercial feedlot cattle harvested in the summer (>80% positive), with EHEC O145, O45, O103, and O157, in descending order, the most prevalent. The results also suggest that at least 6 per 100 carcasses, prior to evisceration and interventions, are contaminated with EHEC-7, based on NS, with EHEC O157, O145, O103, and O45, in descending order, also the most prevalent.

Few studies have estimated the prevalence specifically of EHEC-6 and EHEC-7 on hides and carcasses, as compared to that for E. coli O157:H7 and non-O157 STEC. In one recent study, hide and carcass surface samples were cultured for EHEC-6 and −7, but no isolates were obtained from the hides; in addition, it was unclear whether carcass isolates were EHEC-7 (Svoboda et al., 2013), and the methods yielded only presumptive positive results. In our study, GDS yielded a higher proportion of presumptive positives for EHEC-6 on carcasses than the proportion of confirmed positive samples obtained using NS, and no isolates were obtained from these samples. Prevalence estimates made solely on the detection of O-group plus stx and eae in an enrichment broth sample (presumptive positive) will likely be higher due to false-positive results generated by background organisms containing some, but not all of the gene targets. NS results are reported as confirmed positive or negative; however, recovery of fully characterized bacterial isolates provides the most definitive evidence of a true positive sample. In the present study, GDS testing was not conducted on the hide samples due to the unavailability of the test system in the lab handling these samples.

In contrast to non-O157 EHEC, a relatively large number of studies have reported on the prevalence of EHEC O157:H7 on hides and pre-evisceration beef carcass surfaces. A summarization of the results from these studies is provided in Table 5. Varying methods, media, geographical locations, and seasons undoubtedly contribute to differences in reported prevalence. In the present study, only 6 colonies per plate for each of the 7 IMS serogroups (i.e., 42 colonies per sample) were picked, but the chromogenic phenotype was shared by many of the background flora; hence this protocol is relatively insensitive. Interestingly, the culture-based protocol used in the study by Arthur et al. (2002) yielded similar reported EHEC-6 prevalence levels.

Table 5.

Previous Studies on the Prevalence of Enterohemorrhagic Escherichia coli O157:H7 and “Top 6” Non-O157 on Hides and De-hided Pre-evisceration Carcasses as Detected by Culture-Based Methods

Prevalence
O157%	“Top 6” non-O157 %	Season	Sample area	Sample type	Enrichment	Immunomagnetic separation	Plating	Reference
13.0	NA	Summer	450 cm²	Hide	2% BGB, 37°C 6 h	O157	CT-SMAC	Elder et al., 2000
43.4	NA	Summer	4000 cm²	Carcass	2% BGB, 37°C 10 h	O157	CT-SMAC and BCM
NA	1.2	Summer	4000 cm²	Carcass	2% BGB, 37°C 10 h	No	TSA, colony hybridization for stx followed by SMAC	Arthur et al., 2002
60.6	ND	Year-round	1700 cm²	Hide	TSB, 25°C 2 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow	Barkocy-Gallagher et al., 2003
73.8	ND	Spring	1700 cm²	Hide	TSB, 25°C 2 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow
73.5	ND	Summer	1700 cm²	Hide	TSB, 25°C 2 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow
67.2	ND	Fall	1700 cm²	Hide	TSB, 25°C 2 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow
29.4	ND	Winter	1700 cm²	Hide	TSB, 25°C 2 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow
26.7	ND	Year-round	Unknown	Carcass	TSB, 25°C 2 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow
30.3	ND	Spring	Unknown	Carcass	TSB, 25°C 2 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow
31.9	ND	Summer	Unknown	Carcass	TSB, 25°C 2 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow
33.0	ND	Fall	Unknown	Carcass	TSB, 25°C 2 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow
32.9	ND	Winter	Unknown	Carcass	TSB, 25°C 2 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow
88.3	NA	Summer	2000 cm²	Hide	TSB, 25°C 3 h, 42°C 6 h	O157	CT-SMAC and N-Rainbow	Nou et al., 2003
50.0	NA	Summer	8000 cm²	Carcass	TSB, 25°C 3 h, 42°C 6 h	O157	CT-SMAC and N-Rainbow
75.7	NA	Fall	100 cm²	Hide	TSB, 25°C 2 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow	Arthur et al., 2004
14.7	NA	Fall	8000 cm²	Carcass	TSB, 25°C 2 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow
61.9	NA	Spring to Fall	1700 cm²	Hide	TSB, 25°C 2-3 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow	Rivera-Betancourt et al., 2004
7.0	NA	Spring to Fall	4000 cm²	Carcass	TSB, 25°C 2-3 h, 42°C 6 h	O157	CT-SMAC and NT-Rainbow
14.7	NA	Unknown	300 cm²	Hide	BGB, 37°C 6 h	O157	CT-SMAC and T-CO157	Woerner et al., 2006
10.1	NA	Unknown	300 cm²	Carcass	BGB, 37°C 6 h	O157	CT-SMAC and T-CO157
46.9	ND	Year-round	1000 cm²	Hide	TSB, 25°C 2 h, 42°C 6h	O157	CT-SMAC and NT-CO157	Brichta-Harhay et al., 2008
43.2	ND	Spring	1000 cm²	Hide	TSB, 25°C 2 h, 42°C 6h	O157	CT-SMAC and NT-CO157
55.7	ND	Summer	1000 cm²	Hide	TSB, 25°C 2 h, 42°C 6h	O157	CT-SMAC and NT-CO157
38.7	ND	Fall	1000 cm²	Hide	TSB, 25°C 2 h, 42°C 6h	O157	CT-SMAC and NT-CO157
50.4	ND	Winter	1000 cm²	Hide	TSB, 25°C 2 h, 42°C 6h	O157	CT-SMAC and NT-CO157
16.7	ND	Year-round	8000 cm²	Carcass	TSB, 25°C 2 h, 42°C 6h	O157	CT-SMAC and NT-CO157
14.2	ND	Spring	8000 cm²	Carcass	TSB, 25°C 2 h, 42°C 6h	O157	CT-SMAC and NT-CO157
18.3	ND	Summer	8000 cm²	Carcass	TSB, 25°C 2 h, 42°C 6h	O157	CT-SMAC and NT-CO157
14.9	ND	Fall	8000 cm²	Carcass	TSB, 25°C 2 h, 42°C 6h	O157	CT-SMAC and NT-CO157
19.5	ND	Winter	8000 cm²	Carcass	TSB, 25°C 2 h, 42°C 6h	O157	CT-SMAC and NT-CO157
NA	0.0	Year-round	400 cm²	Hide	mTSB+N, 41.5°C 24 h	O157	CT-SMAC	Wieczorek et al., 2011
NA	0.0	Year-round	400 cm²	Carcass	mTSB+N, 41.5°C 24 h	O157	CT-SMAC
NA	0.0	Year-round	100 cm²	Hide	TSB+V, 37°C overnight	No	TBX+SS	Monaghan et al., 2012
NA	0.4	Year-round	Whole carcass	Carcass	TSB+V, 37°C overnight	No	TBX+SS
17.9	0.2	Year-round	100 cm²	Hide	mTSB+CV, 37°C 6 h	O157, O26, O103, O111 and O145	CT-SMAC and C-CCV, followed by EMB agar	Thomas et al., 2012
0.7	0.0	Year-round	100 cm²	Carcass	mTSB, 37°C 6 h	O157, O26, O103, O111 and O145	CT-SMAC and C-CCV, followed by EMB agar

BGB, brilliant green bile broth; TSB, trypticase soy broth; mTSB, trypticase soy broth modified with the addition of bile salts No. 3; N, novobiocin; V, vancomycin; C, cefixime; NA, not applicable; ND, not determined; CT-SMAC, MacConkey agar with sorbitol, cefixime and potassium tellurite; BCM, Biosynth chromogenic media; TSA, trypticase soy agar; SMAC, MacConkey agar with sorbitol; NT-Rainbow, Rainbow^® agar with novobiocin and potassium tellurite; N-Rainbow, Rainbow^® agar with novobiocin; T-CO157, CHROMagar™ O157 with potassium tellurite; NT-CO157, CHROMagar™ O157 with novobiocin and potassium tellurite; TBX+SS, tryptone bile x-glucuronide agar with streptomycin sulphate and sulfamethazine; C-CCV, Chromocult^® coliform agar with cefixime, cefsulodin sodium salt, and vancomycin; EMB, eosin methylene blue agar.

E. coli O157:H7 was isolated from fewer hide samples in this study than in previous ones (Elder et al., 2000; Brichta-Harhay et al., 2008). This may have been the result of using mPossé agar, which in its original formulation was intended for detection of E. coli O26, O103, O111, and O145, but not O157:H7 (Possé et al., 2008). We chose this medium because we had found in preliminary experiments with pure cultures that all seven EHEC serogroups could be screened for in three colony colors. A potential contributing factor to the lower proportion of EHEC O157 detected could have been the prior treatment of cattle with a commercial bacteriophage solution that is lytic for E. coli O157:H7. Improvements in culture-based methods are likely needed for isolation of EHEC, given the disparities we found between detection protocols. However, it should be noted that our culture method detected EHEC-7 in five hide samples that were negative for the respective organism by NS. In these samples, the signals targeted in the NS test were below the level of detection (E. Hosking, personal communication). In addition to providing backup for more sensitive methods, culture-based tests remain important for trace-back of contaminated foods, the identification of novel genes, and other uses as they produce isolates that can be further confirmed and characterized.

Using the MRA developed by Coombes et al. (2008), all EHEC isolates from hide samples in the present study were seropathotype A or B, and had the virulence gene content (e.g., nle and other) expected for this classification. One isolate (serotype O145:[H28]) would have been less likely to cause a disease outbreak, since it lacked complete O-Islands 36, 57, 71, and 122, and contained only 7 of the 16 genes tested for in the MRA. In general, the MRA tests for genes on these four O-Islands that encode effectors that reduce pro-inflammatory signaling (viz., NleB, NleC, NleD, NleH1, and NleH2) and sustain colonization by preventing bacterial detachment and death of infected host epithelial cells (viz., NleD, NleH1, and NleH2; Vossenkämper et al., 2011). In addition, we tested for three other genes on O-Island 122 (efa1, sen, and pagC; Karmali et al., 2003). Since all isolates had efa1 and sen, and all O157:H7, but none of the non-O157 isolates had pagC, we hypothesize that isolates expressing the products of these genes might have increased virulence through enhancement of intestinal colonization (Efa1), water loss (Sen), and enhanced survival in macrophages (PagC; Karmali et al., 2003; Kitagawa et al., 2010).

Conclusions

The prevalence of enterohemorrhagic E. coli (EHEC) O26, O45, O103, O111, O121, O145, and O157 (EHEC-7) on hides and preintervention de-hided carcasses was estimated from a population of commercial feedlot cattle at harvest. The prevalence of EHEC-7 was 1.2% on hides based on culture, but 80.7% on hides and 6.0% on carcasses based on the NeoSEEK^™ STEC Detection and Identification Test (NS). The prevalence of EHEC-7 on hides was significantly greater than that on carcasses, based on these NS results. This study provides evidence that “top 6” non-O157 and O157:H7 EHEC are present on the hides and carcasses of commercial feedlot cattle at harvest; however, continued improvement of methods is needed for accurate detection and estimation of non-O157 EHEC prevalence, given the discordant results across protocols.

Footnotes

Acknowledgments

We thank David Hanks, Megan Spencer, Diana Dewsbury, Donka Milke, Anteelah Phebus, and Rachael Henderson for technical assistance, Dr. Mick Bosilevac for comments on the data, and Dr. T.G. Nagaraja and Xiaorong Shi for assistance with molecular analysis of samples and isolates. This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2012-68003-30155 from the USDA National Institute of Food and Agriculture.

Disclosure Statement

No competing financial interests exist.

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Prevalence of Enterohemorrhagic Escherichia coli O26,O45,O103,O111,O121,O145,and O157 on Hides and Preintervention Carcass Surfaces of Feedlot Cattle at Harvest

Abstract

Introduction

Materials and Methods

Study design and sample collection

Culture of hide and carcass samples

Detection and characterization of EHEC isolates from hide samples

Detection of EHEC by the NeoSEEK™ STEC Detection and Identification (NS) test

Statistical analysis

Results

Detection of EHEC in hide sponge samples by culture-based and multiplex PCR assays

Characterization of EHEC-7 hide isolates

Detection of EHEC on hides and carcasses by NS

Model-adjusted cumulative prevalence estimates of EHEC on hides and carcasses

Comparison of culture-based method versus NS for EHEC-7 detection in hide samples and GDS versus NS for carcass samples

Discussion

Conclusions

Footnotes

Acknowledgments

Disclosure Statement

References

Detection of EHEC by the NeoSEEK^™ STEC Detection and Identification (NS) test