Culture-Based Quantification with Molecular Characterization of Non-O157 and O157 Enterohemorrhagic Escherichia coli Isolates from Rectoanal Mucosal Swabs of Feedlot Cattle

Abstract

Enterohemorrhagic Escherichia coli (EHEC) strains are foodborne pathogens carried in the intestinal tracts of ruminants and shed in the feces. High concentrations (≥10⁴ colony-forming units [CFU]/g) of EHEC in cattle feces are associated with contamination of hides, and subsequently, carcasses and beef. Several studies using agar media have quantified O157 but few have quantified non-O157 EHEC in samples from cattle. Thus, the objective of this study was to determine the concentration of O157 and non-O157 EHEC in cattle, and to characterize the associated EHEC isolates for their virulence potential. Two hundred feedlot steers were sampled by rectoanal mucosal swab (RAMS) every 35 days over four sampling periods, and a spiral plating method using modified Possé differential agar was used to quantify EHEC organisms in these samples. Bacterial colonies from agar plates were tested by multiplex PCR for Shiga toxin and intimin genes (stx and eae, respectively), and confirmed EHEC isolates (i.e., positive for both stx and eae) were serotyped and characterized for virulence genes using a microarray. Organisms detected in this study included O26, O101, O103, O109, O121, O145, O157, and O177 EHEC, with all except O121 quantifiable and measuring within a range from 9.0 × 10² to 3.0 × 10⁵ CFU/g of RAMS sample. Organisms of the same EHEC serogroup were not detected in quantifiable concentrations from a single animal more than once. EHEC organisms most commonly detected at quantifiable levels were O26, O157, and O177. Interestingly, O26 EHEC isolates tested negative for stx ₁ but positive for stx _2a. High concentrations of EHEC were detected in 11 (5.5%) of the steers at least once over the sampling period. These results indicate that in addition to O157, non-O157 EHEC are transiently present in high concentrations in the rectoanal mucosal region of cattle.

Introduction

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen commonly carried in the intestinal tracts of cattle and shed in their feces. In addition to carrying genes for Shiga toxin (stx), typical EHEC strains possess genes for intimin (eae), which allows intestinal adherence when expressed (Croxen et al., 2013). EHEC concentration in cattle feces is directly associated with hide and carcass contamination (Arthur et al., 2010), and therefore a critical factor to assess when evaluating risk of microbial contamination of beef. Recently, the U.S. Department of Agriculture Food Safety and Inspection Service declared O26, O45, O103, O111, O121, O145, and O157 EHEC (collectively termed EHEC-7) adulterants in nonintact raw beef products (USDA FSIS, 2011).

Nucleic acid-based methods have been developed to quantify non-O157 EHEC in cattle feces (Luedtke et al., 2014; Shridhar et al., 2016), but they have limitations. Multiplex PCR assays use multiple targets to determine whether genes for specific O antigens, stx, and eae are present in a sample, but do not determine whether these genes are contributed by only one or more than one organism. In contrast, isolation on agar media ensures that these gene targets originate from a single organism. A spiral plating method was reliable for quantification of O157 EHEC, and reduced processing time compared to spread-plate methods (Robinson et al., 2004). Previous studies using culture-based methods to determine EHEC concentration in cattle have focused on O157, with few studies on non-O157 organisms (Berry and Wells, 2008; Stromberg et al., 2016a; Shridhar et al., 2017a).

Cattle shedding O157:H7 EHEC at high concentrations (≥10⁴ CFU/g of feces) greatly contribute to the spread of this pathogen between animals in the feedlot (Arthur et al., 2010), and may pose a threat to human health (Chase-Topping et al., 2008). Furthermore, O103 EHEC has been found at high concentrations in cattle feces (Shridhar et al., 2017a). The terminal rectum is a major colonization site of O157:H7 EHEC in cattle and rectoanal mucosal swabs (RAMS) of this region are a convenient and sensitive method of sampling for detection of both O157 and non-O157 EHEC (Agga et al., 2017). It is unclear whether non-O157 EHEC organisms are carried or shed in high concentrations, and if so, how often. In addition to concentration, virulence is important for determining risk. Thus, the objectives of this study were to determine the concentrations of O157 and non-O157 EHEC in RAMS, and to identify the virulence characteristics of quantifiable EHEC isolates in these samples from feedlot steers.

Materials and Methods

Research animals

Two hundred steers (castrated male cattle) at the University of Nebraska-Lincoln Eastern Nebraska Research and Extension feedlot (Ithaca, NE), 13–15 months at the time of first sampling, were included in this study. The steers represented block 1 of another study that included four blocks, each with 200 steers that assessed the effects of diet on EHEC prevalence using a sensitive molecular detection method (Schneider et al., 2017). Steers were assigned randomly, 8 per pen, to 25 feedlot pens in a balanced manner to different corn-based diets. As noted above, an objective of the present study was to determine EHEC concentrations in RAMS samples. The effects of diet on concentration were not determined since the relatively low sample number (n = 200) and prevalence across EHEC serogroups precluded adequate hypothesis testing. All procedures involving steer handling and sampling were approved by the UNL Institutional Animal Care and Use Committee.

Sample collection and processing

RAMS samples were taken from the steers once every 35 days over four sampling periods, with the last sample ∼30 days before harvest: May 19, June 23, July 28, and September 1, 2015. One steer was removed before the fourth sampling period due to chronic bloat; hence, 799 RAMS samples were obtained. To obtain RAMS samples, each individual steer was restrained in a squeeze chute, a foam-tipped swab (VWR International, Buffalo Grove, IL) was inserted 3–5 cm into the terminal rectum, and the mucosal surface was swabbed.

Immediately after RAMS collection, the sample contents, estimated as 0.5 g, were transferred into 15-mL conical tubes containing 5 mL chilled (5°C) EC broth (Oxoid, Basingstoke, United Kingdom). Conical tubes containing the samples were held in Styrofoam racks (50 tubes/rack) surrounded on all sides by frozen, reusable freezer packs (n = 8–10) in a 45-L cooler (30.5 × 58.4 × 25.6 cm). The RAMS samples (all in one cooler per sampling, n = 200) were delivered to the laboratory within 3.5 h after collection, and maintained at ∼8.5°C. Upon arrival at the laboratory, tubes were vigorously mixed by vortexing for 10 s and contents allowed to settle for 1 min. A 1-mL aliquot was removed and frozen in 0.5 mL of BBL™ brain heart infusion (Becton, Dickinson and Company, Sparks, MD) broth with 50% glycerol at −80°C within 2.5 h after the RAMS samples arrived at the laboratory (i.e., within 6 h after sample collection). Hence, samples were near or below the minimum cardinal temperature of E. coli (Van Derlinden et al., 2008) until cultured.

Quantification of EHEC

Samples stored at −80°C were allowed to thaw at room temperature for 2 h. Using a spiral plater (Eddy Jet 2; IUL Instruments, Königswinter, Germany), 50 μL of the culture was spiral plated onto a modification of Possé agar containing 5.0 mg/L novobiocin and 0.5 mg/L potassium tellurite as previously described (Stromberg et al., 2015). Plates were incubated at 37°C for 18 h. After incubation, blue-purple, red-purple, and green colonies were quantified as potential EHEC. Colonies (≤10/plate) of these target phenotypes were randomly picked in approximately equal numbers and heated at 95°C in 50 μL of water for use as DNA template. Colonies were tested for EHEC-7 serogroups, stx ₁, stx ₂, eae, and EHEC-hemolysin (ehxA) by an 11-plex PCR assay (Stromberg et al., 2016a). The concentration of EHEC in the sample was determined based on the proportion of target colonies on an individual plate confirmed to be EHEC organisms. The theoretical limit of quantification (LoQ) of the spiral plate assay was 7.5 × 10² CFU/g of feces (Maturin and Peeler, 2001). However, a validation study of the assay conducted exactly as described herein, with the exception of a volume of 100 μL being plated instead of 50 μL, indicated the LoQ is 10³–10⁵ CFU/g depending on O-group (Shridhar et al., 2017a).

Characterization of EHEC isolates

Isolates that tested positive for stx and eae, but not identified as an EHEC-7 serogroup were serotyped at The Pennsylvania State University, E. coli Reference Center. Isolates were further characterized by the U.S. Food and Drug Administration E. coli Identification Microarray (FDA-ECID) for putative virulence-associated genes and to determine whether EHEC isolates were genetically identical (Patel et al., 2016).

Results

Concentration of EHEC in fecal samples

EHEC concentration results are summarized in Table 1. Of 799 RAMS samples, 44 (5.5%) were positive for EHEC, 27 (3.4%) were EHEC-7 positive including serogroups O26, O103, O121, O145, and O157, and 18 (2.3%) were positive for other non-O157 EHEC (O101, O109, and O177). Of the 200 steers sampled, 40 were EHEC positive at one or more periods during the study. Twenty-one, 18, 2 and 4 steers were EHEC positive on days 0, 35, 70, and 105, respectively. Only three steers were positive for EHEC at multiple time points, and none shed the same EHEC serogroup across sampling periods. Of the quantifiable samples, concentration values ranged from 9.0 × 10² to 3.0 × 10⁵ CFU/g of feces. Eleven (5.5%) steers from eight different pens were high-level shedders (≥10⁴ CFU/g) at some time point. No pens contained more than one steer shedding the same EHEC serogroup at high levels during a single sampling period. In one pen, a steer shed high-levels of O177 EHEC (profile 14) at sample period 2 (June sampling), and a different steer from the same pen shed O177 EHEC (profile 14) at high-concentrations at period 4 (September sampling). This was the only pen that had steers shedding the same serogroup at levels ≥10⁴ CFU/g for more than one sampling date.

Table 1.

Concentration of Enterohemorrhagic Escherichia coli in Cattle Rectoanal Mucosal Swabs

	No. of samples positive ^a
Serotype	<750 CFU/g	10 ² CFU/g	10 ³ CFU/g	10 ⁴ CFU/g	10 ⁵ CFU/g	Total ^b
EHEC O26:H11	3	0	6	2	1	12
EHEC O101:H33	0	0	1	0	0	1
EHEC O103:H2	0	0	1	0	0	1
EHEC O109:H10	1	1	4	1	0	7
EHEC O121:H19	1	0	0	0	0	1
EHEC O145:H28	1	0	1	1	0	3
EHEC O157:H7	1	0	7	2	0	10
EHEC O177:H25	1	0	5	3	1	10
Total	8	1	25	9	2	44^b

Values below 7.5 × 10² CFU/g represent a sample in which an EHEC organism was detected by the assay below the limit of quantification. Values of 10² CFU/g were samples quantified between 7.5 × 10² and 9.9 × 10² CFU/g.

One sample contained quantifiable levels of both O26 and O109 EHEC organisms; hence, a total of 44 instead of 45 samples were EHEC positive.

EHEC, enterohemorrhagic E. coli.

Shiga toxin subtypes of EHEC isolates

Isolates were characterized by the FDA-ECID microarray (Table 2). All EHEC isolate serotypes were confirmed by independent tests conducted at the E. coli Reference Center (The Pennsylvania State University). Isolates were further characterized for Shiga toxin subtypes. The stx _1a gene was found in O103 and O145 EHEC isolates, and all except one O157 EHEC isolate were positive for stx _1a, stx _2a, stx _2c, and stx _2d. All O177 EHEC along with one O157 EHEC isolate were positive for stx _2a, stx _2c, and stx _2d. All O26, O101, O109, and O121 EHEC were positive for stx _2a.

Table 2.

Molecular Characterization Of Enterohemorrhagic Escherichia coli Isolates

				Adhesin				Effector				Serine protease		T3SS ^c			Toxin
Serotype	Month (no. isolated)	Animal ID ^a	ECID profile ^b	eae	efa1	fimH	toxB	nleB	nleC	nleH	tir	eaaA	eatA	escD	escN	escR	ehxA	sta1	stx₁	stx₂
O26:H11	May (11), June (1)	5741, 5742, 5832, 6487, 6788, 7004, 7015, 7040, 7174, 7200, 7225, 7459	1	β1	+	+	+	–	–	+	+	+	+	+	+	+	+	–	–	a
O101:H33	June (1)	7720	2	ι1	–	–	–	–	–	–	–	+	+	–	–	–	+	–	–	a
O103:H2	May (1)	6803	3	ɛ	+	+	–	+	+	+	+	–	–	+	+	+	+	–	a	–
O109:H10	May (1), June (10)	5787, 6666, 6852, 7004, 7242, 7459, 7559	4	ι1	–	+	–	–	–	–	+	–	+	+	–	+	+	+	–	a
O109:H10	June (1)	7459	5	ι1	–	+	–	–	–	–	–	–	+	+	–	+	+	+	–	a
O109:H10	June (1)	6666	6	ι1	–	+	–	–	–	–	–	–	+	+	–	+	+	+	–	a
O121:H19	May (1)	6832	7	ɛ	+	+	–	–	–	–	+	–	+	+	+	–	+	–	–	a
O145:H28	June (1), July (1)	5792, 7806	8	γ1	–	+	–	+	+	+	–	–	+	+	–	–	+	–	a	–
O145:H28	June (1)	7721	9	γ1	–	+	–	+	+	+	–	–	+	+	–	–	+	–	a	–
O157:H7	May (5), June (1)	5792, 6369, 6918, 7452, 7453, 7460	10	γ1	–	+	+	+	+	+	+	–	–	+	–	+	+	–	a	acd
O157:H7	June (1)	7823	11	γ1	–	+	+	+	+	+	+	–	–	+	–	+	+	–	a	–
O157:H7	June (2), July (1)	5638, 5832, 7162	12	γ1	–	+	+	+	+	+	+	–	–	+	–	+	+	–	a	acd
O177:H25	May (1)	7208	13	β1	–	–	–	–	–	+	+	–	+	+	+	–	+	–	–	acd
O177:H25	May (1), June (8), September (9)	5759, 6832, 6959, 6978, 7507, 7649, 7731, 7850	14	β1	–	–	–	–	–	+	+	–	+	+	+	–	+	–	–	acd
O177:H25	June (1)	6514	15	β1	–	+	–	–	–	+	+	–	+	+	+	–	+	–	–	acd

Each animal was given a unique four-digit identification number. Identification numbers in bold represent animals that tested positive for EHEC more than once.

Isolates with the same U.S. Food and Drug Administration E. coli Identification Microarray profile are genetically identical.

T3SS, type III secretion system.

Accessory virulence-associated genes

EHEC isolates were characterized for the presence of additional virulence-associated genes by the FDA-ECID microarray (Table 2). For adherence factors, the EHEC factor for adherence (efa1) was detected in 14 (23%) isolates, the type 1 fimbrial adhesin gene (fimH) in 41 (67%), and the adherence-associated toxB gene in 22 (36%). Nonlocus of enterocyte effacement encoded effector (nle) genes nleB, nleC, and nleH were detected in 14 (23%), 14 (23%), and 46 (75%) isolates, respectively. The translocated intimin receptor gene (tir) was detected in 24 (39%) isolates. Serine protease autotransporter genes, namely eaaA and eatA, were detected in 13 (21%) and 50 (82%) of the isolates. Type III secretion system genes escD, escN, and escR were detected in 60 (98%), 34 (56%), and 36 (59%) isolates, respectively. Only O109 EHEC isolates were positive for the heat-stable enterotoxin gene, sta1.

Comparison of genetic relatedness between EHEC isolates

Isolates were compared for genetic relatedness as determined by the FDA-ECID microarray (Table 2). For the different non-O157 EHEC isolates, the number found to be the same strains were 13 of 13 O26; 11 of 13 O109; 2 of 3 O145; and 18 of 20 O177. Of the O101, O103, and O121 serogroups, only one isolate each was detected. Six of nine O157 EHEC isolates were the same strain, and three others were another strain.

Discussion

EHEC colonizes the intestine of cattle and remains a risk to food safety through contamination of beef products during processing (Croxen et al., 2013). At slaughter, concentration may be a greater risk factor than prevalence for transmission of EHEC from feces to hides, and subsequently onto the carcass (Arthur et al., 2010). Previous studies have determined the prevalence, but few have quantified both O157 and non-O157 EHEC in samples collected from cattle (Luedtke and Bosilevac, 2015; Stromberg et al., 2016a). Molecular assays were developed to quantify EHEC in cattle feces (Luedtke et al., 2014; Verstraete et al., 2014; Noll et al., 2015; Shridhar et al., 2016), but in a polymicrobial matrix, multiple organisms could contribute one or more target genes leading to a false positive result. In the current study using culture-based quantification, O26 was the most frequently isolated EHEC serogroup followed by O157, O177, O109, O145, O101, O103, and O121. O26 EHEC strains are frequently isolated from cattle feces (Pearce et al., 2006; Stanford et al., 2016), and this EHEC serogroup accounts for most non-O157 EHEC clinical infections in Europe and the U.S. (Bielaszewska et al., 2013). Since there is no gold standard method to detect non-O157 EHEC in feces, the agar medium used in this study may have been better suited for those EHEC serogroups it was originally developed to isolate, including O26 (Possé et al., 2008), with resultant infrequent detection of others.

In previous studies, most cattle fecal samples with quantifiable levels contained <10⁴ CFU O157 EHEC/g (Omisakin et al., 2003; Brichta-Harhay et al., 2007; Arthur et al., 2010). In a study of concentration of O157 EHEC in cattle feces at slaughter, 75% and 91% contained <10³ and <10⁴ CFU/g, respectively (Omisakin et al., 2003). Brichta-Harhay et al. (2007) reported that 71% of samples contained O157 EHEC at <10⁴ CFU/g of cattle feces using an agar specific for E. coli O157:H7. This study also found 16% of samples contained >10⁵ CFU O157 EHEC/g, and one sample contained >10⁶ CFU/g (Brichta-Harhay et al., 2007). Of the enumerated fecal samples in the current study, 80% (8/10) had <10⁴ CFU O157 EHEC/g, which was similar to aforementioned studies. However, we found previously that use of agar media designed to isolate multiple EHEC serogroups underestimated the actual concentration of EHEC from inoculated cattle feces (Stromberg et al., 2016b). In a complex sample such as feces, background organisms may present a challenge to assess the true concentration of EHEC. In addition, increasing the number of suspect colonies tested would likely increase the sensitivity of the assay, but based on technical feasibility, only 10 suspect colonies were tested per sample.

Few studies have assessed the concentration of non-O157 EHEC in cattle feces. Herein, most fecal samples were positive for only one EHEC serogroup, except one sample that contained quantifiable levels of both O26 and O109 EHEC. A total of 74% of the fecal samples contained <10⁴ CFU non-O157 EHEC/g, which was similar to levels detected for O157 EHEC. Luedtke et al. (2014) used a molecular assay to quantify total EHEC load in cattle feces and found 7 × 10¹ to 3 × 10⁵ CFU/ml, comparable to that in the current study. Shridhar et al. (2017a) used a similar spiral plating method and found only one non-O157 EHEC (O103) quantifiable in only one of 1,152 cattle fecal samples. Similarly, the current study detected only one quantifiable EHEC O103 in only one of 799 RAMS samples, but contrary to Shridhar et al. (2017a), other non-O157 EHEC were present at quantifiable concentrations. A separate study in Irish dairy herds, found cattle shed high concentrations of O26 EHEC (Murphy et al., 2016). Comparably, we quantified O26 EHEC in RAMS samples at concentrations >10⁴ CFU/g.

EHEC isolates were characterized for virulence factors including stx subtype by a microarray (FDA-ECID) that tests for >40,000 known E. coli genic regions (Patel et al., 2016). All O26, O101, O109, and O121 EHEC isolates were positive for stx _2a. These same EHEC serogroups were associated with human disease in previous studies (Bettelheim, 2007). Additionally, O26 and O121 EHEC are adulterants in raw non-intact beef according to the USDA-FSIS (USDA, 2011). Typically, O26 EHEC isolated from cattle and clinical cases are stx ₁-positive, but O26 EHEC stx _2a-positive organisms have been isolated from cattle and there has been an increase in severe clinical cases associated with these organisms (Bielaszewska et al., 2013; Zweifel et al., 2013). In a separate study determining risk associated with EHEC virulence factors, EHEC strains carrying stx _2a showed a strong association with hemolytic uremic syndrome (HUS) in human patients (Brandal et al., 2015) and greater likelihood of HUS than EHEC strains carrying stx ₁ instead. Furthermore, Stx2a protein is highly toxic in vitro to human renal proximal tubule epithelial cells, and in vivo in mice (Fuller et al., 2011). Previous selected studies have found a range of 16–42% of Shiga toxin-producing E. coli isolates from cattle possessed stx _2a (Chui et al., 2015; Mellor et al., 2015; Shridhar et al., 2017b), which was similar to the proportion of EHEC isolates testing positive for stx _2a in the current study (44%). Collectively, these results suggest that cattle serve as a source of O26 EHEC carrying stx _2a.

Other non-O157 EHEC such as O101, O109, and O177 have been isolated from healthy cattle and human patients (Bettelheim, 2007). O109 stx _2a-positive EHEC have been isolated from cattle (Akiyama et al., 2017). In addition, O177 EHEC were detected on pre-evisceration veal carcasses and in feces and hides of culled dairy cows at harvest (Stromberg et al., 2016a; Bosilevac et al., 2017). Currently, these organisms are not adulterants in raw, nonintact beef according to the USDA-FSIS, but still pose a potential threat to human health.

The virulence of O157:H7 EHEC is well documented, but virulence potential for certain non-O157 EHEC remains unclear. O26 and O103 EHEC isolates contained virulence genes suggesting they were capable of causing attaching and effacing (A/E) lesion formation, for example, eae, tir, and type III secretion system genes (escD, escN, and escR). These isolates also contained additional adherence factor genes (efa1, fimH, and toxB), the encoded products of which may contribute to intestinal adherence (Farfan and Torres, 2012). O121 and O177 EHEC shared most of these same virulence genes, but lacked escR and those for some additional adherence factors. Conversely, O101 EHEC lacked the same type III secretion system genes, tir, and genes for additional adherence factors, and thereby lacked the ability to cause A/E lesions. O109 EHEC isolates were positive for two of three type III secretion system genes, but only one isolate was positive for tir. Similarly, O145 EHEC isolates in this study presumably lack the ability to form A/E lesions due to the absence of tir. This suggests that of the non-O157 EHEC isolates, only those belonging to serogroups O26, O103, O121, and O177 EHEC would have the potential to cause A/E lesions.

In addition to molecular characterization, the isolates in this study were analyzed with the FDA-ECID to determine whether different animals were shedding the same strain. Within serogroup, most EHEC isolates were genetically identical indicating that different animals were shedding the same strain, but at different concentrations. Although genotypic differences of EHEC appear to play a role in high level shedding (Cote et al., 2015), the same EHEC strains can be shed by different animals at various concentrations (Munns et al., 2016).

In summary, this study assessed EHEC concentration in RAMS from steers using a culture-based quantification method. EHEC serogroups commonly found in human clinical cases were present and other potentially pathogenic non-O157 EHEC. O157 and non-O157 EHEC strains were transiently present at quantifiable levels and in a minority of animals at high concentrations (≥10⁴ CFU/g). This work further underscores the continued need for development and validation of effective interventions for EHEC in cattle, both pre- and postharvest.

Footnotes

Acknowledgments

This material is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award 2012-68003-30155. The authors thank Ms. HaNa Kwon for technical assistance.

Disclosure Statement

No competing financial interests exist.

References

Agga

, Arthur

, Hinkley

, Bosilevac

. Evaluation of rectoanal mucosal swab sampling for molecular detection of enterohemorrhagic Escherichia coli in beef cattle. J Food Prot, 2017; 80:661–667.

Akiyama

, Futai

, Saito

, Ogita

, Sakae

, Fukunaga

, Tsuji

, Chikahira

, Iguchi

. Shiga toxin subtypes and virulence genes in Escherichia coli isolated from cattle. Jpn J Infect Dis, 2017; 70:181–185.

Arthur

, Britchta-Harhay

, Bosilevac

, Kalchayanand

, Shackelford

, Wheeler

, Koohmaraie

. Super shedding of Escherichia coli O157:H7 by cattle and the impact on beef carcass contamination. Meat Sci, 2010; 86:32–37.

Berry

, Wells

. A direct plating method for estimating populations of Escherichia coli O157 in bovine manure. J Food Prot, 2008; 71:2233–2238.

Bettelheim

. The non-O157 Shiga-toxigenic (Verocytotoxigenic) Escherichia coli; under-rated pathogens. Crit Rev Microbiol, 2007; 33:67–87.

Bielaszewska

, Mellmann

, Bletz

, Zhang

, Köck

, Kossow

, Prager

, Fruth

, Orth-Höller

, Marejková

, Morabito

, Caprioli

, Piérard

, Smith

, Jenkins

, Curová

, Karch

. Enterohemorrhagic Escherichia coli O26:H11/H-: A new virulent clone emerges in Europe. Clin Infect Dis, 2013; 56:1373–1381.

Brandal

, Wester

, Lange

, Løbersli

, Lindstedt

, Vold

, Kapperud

. Shiga toxin-producing Escherichia coli infections in Norway, 1992–2012: Characterization of isolates and identification of risk factors for haemolytic uremic syndrome. BMC Infect Dis, 2015; 15:324.

Brichta-Harhay

, Arthur

, Bosilevac

, Guerini

, Kalchayanand

, Koohmaraie

. Enumeration of Salmonella and Escherichia coli O157:H7 in ground beef, cattle carcass, hide and faecal samples using direct plating methods. J Appl Microbiol, 2007; 103:1657–1668.

Bosilevac

, Wang

, Luedtke

, Hinkley

, Wheeler

, Koohmaraie

. Characterization of enterohemorrhagic Escherichia coli on veal hides and carcasses. J Food Prot, 2017; 80:136–145.

10.

Chase-Topping

, Gally

, Low

, Matthews

, Woolhouse

. Super-shedding and the link between human infection and livestock carriage of Escherichia coli O157. Nat Rev Microbiol, 2008; 6:904–912.

11.

Chui

, Li

, Fach

, Delannoy

, Malejczyk

, Patterson-Fortin

, Poon

, King

, Simmonds

, Scott

, Lee

. Molecular profiling of Escherichia coli O157:H7 and non-O157 strains isolated from humans and cattle in Alberta, Canada. J Clin Microbiol, 2015; 53:986–990.

12.

Cote

, Katani

, Moreau

, Kudva

, Arthur

, DebRoy

, Mwangi

, Albert

, Raygoza Garay

, Li

, Brandl

, Carter

, Kapur

. Comparative analysis of super-shedder strains of Escherichia coli O157:H7 reveals distinctive genomic features and a strongly aggregative adherent phenotype on bovine rectoanal junction squamous epithelial cells. PLoS One, 2015; 10:e0116743.

13.

Croxen

, Law

, Scholz

, Keeney

, Wlodarska

, Finlay

. Recent advances in understanding enteric pathogenic Escherichia coli . Clin Microbiol Rev, 2013; 26:822–880.

14.

Farfan

, Torres

. Molecular mechanisms that mediate colonization of Shiga toxin-producing Escherichia coli strains. Infect Immun, 2012; 80:903–913.

15.

Fuller

, Pellino

, Flagler

, Strasser

, Weiss

. Shiga toxin subtypes display dramatic differences in potency. Infect Immun, 2011; 79:1329–1337.

16.

Luedtke

, Bono

, Bosilevac

. Evaluation of real time PCR assays for the detection and enumeration of enterohemorrhagic Escherichia coli directly from cattle feces. J Microbiol Methods, 2014; 105:72–79.

17.

Luedtke

, Bosilevac

. Comparison of methods for the enumeration of enterohemorrhagic Escherichia coli from veal hides and carcasses. Front Microbiol, 2015; 6:1062.

18.

Maturin

, Peeler

. Aerobic plate count. In Merker

(ed.): Food and Drug Administration Bacteriological Analytic Manual, 8th Edition. Gaithersburg, Maryland: AOAC International, 2001.

19.

Mellor

, Fegan

, Gobius

, Smith

, Jennison

, D'Astek

, Rivas

, Shringi

, Baker

, Besser

. Geographically distinct Escherichia coli O157 isolates differ by lineage, Shiga toxin genotype, and total Shiga toxin production. J Clin Microbiol, 2015; 53:579–586.

20.

Munns

, Zaheer

, Xu

, Stanford

, Laing

, Gannon

, Selinger

, McAllister

. Comparative genomic analysis of Escherichia coli O157:H7 isolated from super-shedder and low-shedder cattle. PLoS One, 2016; 11:e0151673.

21.

Murphy

, McCabe

, Murphy

, Buckley

, Crowley

, Fanning

, Duffy

. Longitudinal study of two dairy herds: Low numbers of Shiga toxin-producing Escherichia coli O157 and O26 super-shedders identified. Front Microbiol, 2016; 7:1850.

22.

Noll

, Shridhar

, Shi

, An

, Cernicchiaro

, Renter

, Nagaraja

, Bai

. A four-plex real-time PCR assay, based on rfbE, stx ₁, stx ₂, and eae genes, for the detection and quantification of Shiga toxin-producing Escherichia coli O157 in cattle feces. Foodborne Pathog Dis, 2015; 12:787–794.

23.

Omisakin

, MacRae

, Ogden

, Strachan

. Concentration and prevalence of Escherichia coli O157 in cattle feces at slaughter. Appl Environ Microbiol, 2003; 69:2444–2447.

24.

Patel

, Gangiredla

, Lacher

, Mammel

, Jackson

, Lampel

, Elkins

. FDA Escherichia coli identification (FDA-ECID) microarray: A pangenome molecular toolbox for serotyping, virulence profiling, molecular epidemiology, and phylogeny. Appl Environ Microbiol, 2016; 82:3384–3394.

25.

Pearce

, Evans

, McKendrick

, Smith

, Knight

, Mellor

, Woolhouse

, Gunn

, Low

. Prevalence and virulence factors of Escherichia coli serogroups O26, O103, O111, and O145 shed by cattle in Scotland. Appl Environ Microbiol, 2006; 72:653–659.

26.

Possé

, De Zutter

, Heyndrickx

, Herman

. Novel differential and confirmation plating media for Shiga toxin-producing Escherichia coli serotypes O26, O103, O111, O145 and sorbitol-positive and -negative O157. FEMS Microbiol Lett, 2008; 282:124–131.

27.

Robinson

, Wright

, Williams

, Hart

, French

. Development and application of a spiral plating method for the enumeration of Escherichia coli O157 in bovine faeces. J Appl Microbiol, 2004; 97:581–589.

28.

Schneider

, Klopfenstein

, Stromberg

, Lewis

, Erickson

, Moxley

, Smith

. A randomized controlled trial to evaluate the effects of dietary fibre from distillers grains on enterohemorrhagic Escherichia coli detection from the rectoanal mucosa and hides of feedlot steers. Zoonoses Public Health, 2017. DOI: 10.1111/zph.12379 [Epub ahead of print].

29.

Shridhar

, Noll

, Cull

, Shi

, Cernicchiaro

, Renter

, Bai

, Nagaraja

. Spiral plating method to quantify the six major non-O157 E. coli serogroups in cattle feces. J Food Prot, 2017a;80:848–856.

30.

Shridhar

, Noll

, Shi

, An

, Cernicchiaro

, Renter

, Nagaraja

, Bai

. Multiplex quantitative PCR assays for the detection and quantification of the six major non-O157 Escherichia coli serogroups in cattle feces. J Food Prot, 2016; 79:66–74.

31.

Shridhar

, Siepker

, Noll

, Shi

, Nagaraja

, Bai

. Shiga toxin subtypes of non-O157 Escherichia coli serogroups isolated from cattle feces. Front Cell Infect Microbiol, 2017b;7:121.

32.

Stanford

, Johnson

, Alexander

, McAllister

, Reuter

. Influence of season and feedlot location on prevalence and virulence factors of seven serogroups of Escherichia coli in feces of Western-Canadian slaughter cattle. PLoS One, 2016; 11:e0159866.

33.

Stromberg

, Baumann

, Lewis

, Sevart

, Cernicchiaro

, Renter

, Marx

, Phebus

, Moxley

. Prevalence of enterohemorrhagic Escherichia coli O26, O45, O103, O111, O121, O145, and O157 on hides and preintervention carcass surfaces of feedlot cattle at harvest. Foodborne Pathog Dis, 2015; 12:631–638.

34.

Stromberg

, Lewis

, Aly

, Lehenbauer

, Bosilevac

, Cernicchiaro

, Moxley

. Prevalence and level of enterohemorrhagic Escherichia coli in culled dairy cows at harvest. J Food Prot, 2016a;79:421–431.

35.

Stromberg

, Lewis

, Moxley

. Comparison of agar media for detection and quantification of Shiga toxin-producing Escherichia coli in cattle feces. J Food Prot, 2016b;79:939–949.

36.

U.S. Department of Agriculture, Food Safety and Inspection Service. Shiga toxin-producing Escherichia coli in certain raw beef products. 9 CFR, parts 416, 417, and 430. Fed Regist, 2011; 76:58157–58165.

37.

Van Derlinden

, Bernaerts

, Van Impe

. Accurate estimation of cardinal growth temperatures of Escherichia coli from optimal dynamic experiments. Int J Food Microbiol, 2008; 128:89–100.

38.

Verstraete

, Van Coillie

, Werbrouck

, Van Weyenberg

, Herman

, Del-Favero

, De Rijk

, De Zutter

, Joris

, Heyndrickx

, De Reu

. A qPCR assay to detect and quantify Shiga toxin-producing E. coli (STEC) in cattle and on farms: A potential predictive tool for STEC culture-positive farms. Toxins, 2014; 6:1201–1221.

39.

Zweifel

, Cernela

, Stephan

. Detection of the emerging Shiga toxin-producing Escherichia coli O26:H11/H- sequence type 29 (ST29) clone in human patients and healthy cattle in Switzerland. Appl Environ Microbiol, 2013; 79:5411–5413.