Influence of Wet Distiller's Grains on Prevalence of Escherichia coli O157:H7 and Salmonella in Feedlot Cattle and Antimicrobial Susceptibility of Generic Escherichia coli Isolates

Abstract

The current research examined the inclusion of 20% wet distiller's grains (WDG) fed with steam-flaked corn (SFC) or dry-rolled corn (DRC) in diets fed to feedlot cattle on fecal prevalence of Escherichia coli O157:H7 and Salmonella. Crossbred beef heifers (n = 272; average initial body weight (BW) = 354 kg) were blocked by BW and pen size and randomly assigned to treatment. Fecal samples from freshly voided fecal pats were collected from each pen on the day cattle shipped for slaughter (237 fecal samples: 72, 125, and 40 from cattle 132, 160, and 181 days on feed, respectively). Fecal samples were cultured quantitatively and qualitatively for the above pathogens. Populations of E. coli O157:H7 and Salmonella were generally low with very few samples containing quantifiable populations. Similarly, after enrichment, few samples were E. coli O157:H7 positive in any collection with no treatment differences (p > 0.10). More samples were Salmonella positive during the first collection with an increased (p < 0.05) prevalence observed in the SFC and DRC treatments compared with DRC + WDG treatment. No other treatment differences were observed for Salmonella. Putative fecal coliform isolates (18 per treatment; first collection) were examined for antimicrobial susceptibility, and the majority were susceptible to all of the antibiotics examined. Most of the resistance was observed in the SFC (n = 3) and DRC (n = 4) treatments, and only one isolate in each of the two WDG treatments demonstrated resistance (one antibiotic each, streptomycin and tetracycline). All multidrug resistance (2–4 antibiotics) was observed in isolates cultured from the DRC and SFC treatments (n = 2 isolates in each treatment). Results of the current research found no significant effect of feeding WDG to feedlot cattle on fecal prevalence, at time of shipment for slaughter, of E. coli O157:H7, and only modest differences (decreases) in Salmonella prevalence with no apparent affect on antimicrobial susceptibility of fecal coliform isolates.

Introduction

F uel ethanol production has expanded rapidly during the past 10 years in the United States, resulting in a surplus of fermentation by-products. These by-products, termed distiller's grains (DG), can be utilized as livestock feed. Not surprisingly, the feeding of DG has increased substantially, particularly in the cattle feeding industry (National Agricultural Statistics Service, 2007). The inclusion of DG has been shown to appreciably affect rumen microbiology and fermentation (Fron et al., 1996). Others have speculated that the relative lack of starch (compared to corn) and the high concentration of fiber and protein escaping ruminal digestion in DG are more likely to influence the hindgut (Jacob et al., 2009). Taken together, these reports suggest that feeding DG, compared with a traditional corn or sorghum grain-based diets, alters the gastrointestinal environment and that it likely influences bacterial populations within the gut. Recent research attempted to determine whether feeding DG influenced fecal prevalence of Escherichia coli O157:H7 and Salmonella in feedlot cattle with mixed results (Jacob et al., 2008b, 2008c, 2009). Previous research reported that feeding grain processed by dry-rolling compared with steam-flaking decreased fecal shedding of E. coli O157:H7 (Fox et al., 2007), whereas others (Jacob et al., 2009) reported no effect of dry-rolled corn (DRC) on fecal prevalence in cattle. Therefore, the first objective of the current research was to evaluate the influence of feeding diets containing either steam-flaked corn (SFC) or DRC with and without 20% wet DG on fecal prevalence of E. coli O157:H7 and Salmonella in feedlot cattle.

Ethanol production utilizes antimicrobials to reduce bacterial contamination, which are thought to be later destroyed during the distillation process (Narendranath et al., 2000). Failure to destroy these antibiotics, or the survival of thermophilic bacteria could potentially serve as a transmission vector for mediators of antimicrobial resistance. The second objective was to examine putative fecal coliform (FC) isolates cultured from cattle fed DG for antimicrobial resistance.

Materials and Methods

Cattle were fed at the Texas AgriLife Research/U.S. Department of Agriculture–Agricultural Research Service joint feeding facilities near Bushland, TX, from November 2006 to May 2007. Crossbred beef heifers (n = 272; average initial body weight [BW] = 354 kg) were blocked by BW and pen size and randomly assigned to treatment. Pens accommodated 8 (4 pens), 10 (16 pens), or 20 head/pen (4 pens). Treatments were arranged in a 2 × 2 factorial and consisted of a corn-processing method (DRC or SFC) and inclusion of 0 or 20% wet DG (WDG) by-products. Other than diet, management of the cattle was similar for all pens and was typical for feedlots in the southwestern United States. Cattle were shipped to slaughter on three separate dates based on final BW after 132, 160, and 181 days on feed (DOF).

Fecal samples from freshly voided fecal pats, free of obvious contamination, were collected from each pen on the day cattle were shipped for slaughter. In most cases we were able to collect the same number of fecal samples from each pen (representing each treatment) on the different samples dates. A total of 237 fecal samples were collected: 72, 125, and 40 from cattle 132, 160, and 181 DOF, respectively. Fecal samples were collected into palpation sleeves, packed on ice, and shipped overnight to our laboratory in College Station, TX, for bacterial culture the following day using methodology described below.

Bacterial culture, isolation, and enumeration methods

All fecal samples were processed the day after collection for qualitative and quantitative analysis of Salmonella (Edrington et al., 2009) and E. coli O157:H7 as described previously (Robinson et al., 2004) and modified (Brichta-Harhay et al., 2007). For purposes of antimicrobial susceptibility screening, putative FCs were selected from chromagar plates based on color and morphology, resuspended in phosphate-buffered saline, and frozen (−80°C) until screening for antimicrobial susceptibility described below. One isolate was randomly selected from each plate from the first collection (132 DOF) for a total of 18 isolates/treatment (72 total coliform isolates). Unless noted otherwise, all reagents and antibiotics were obtained from Sigma Chemical (St. Louis, MO).

Antimicrobial susceptibility was determined on FC isolates using the Sensititre automated antimicrobial susceptibility system and the National Antibiotic Resistance Monitoring System's testing panels (Trek Diagnostic Systems, Cleveland, OH) for gram-negative isolates as described previously (Edrington et al., 2009).

Statistical analysis

Data were analyzed using SAS version 8.02 (SAS, Cary, NC). The percentage of cattle shedding E. coli O157:H7 or Salmonella was subjected to Chi-square analysis using the PROC FREQ procedure and analyzed by day of collection (DOF) and across collections. Differences were considered significant at a 5% level of significance.

Results

Fecal prevalence of E. coli O157:H7 and Salmonella is presented by collection and across collection days in Table 1. Populations of both pathogens were low with only two samples culture positive for E. coli O157:H7 (SFC and SFC + WDG treatments) and one sample positive for Salmonella (SFC + WDG treatment) after direct plating. Two, zero, and seven samples were E. coli O157:H7 positive after immunomagnetic separation in the first, second, and third collections, respectively, with no treatment differences (p > 0.10) observed. More samples were Salmonella positive after a double-enrichment procedure during the first collection with an increased (p < 0.05) prevalence observed in the SFC and DRC treatments compared with DRC + WDG treatment. No other treatment differences were observed for Salmonella at the later collection dates or when data were pooled across dates.

Table 1.

Fecal Prevalence (Percent Positive in Parentheses) of Escherichia coli O157:H7 and Salmonella in Feedlot Cattle Fed Diets Containing Either Steam-Flaked Corn or Dry-Rolled Corn With and Without 20% Wet Distiller's Grains After 132, 160, or 181 Days on Feed

Item	DOF	Method	SFC	SFC + WDG	DRC	DRC + WDG
Escherichia coli O157:H7	132	DIR	0/18	0/18	0/18	0/18
		ENR	1/18 (5.6)	0/18	0/18	1/18 (5.6)
	160	DIR	0/31	0/31	0/31	0/32
		ENR	0/31	0/31	0/31	0/32
	181	DIR	1/10 (10)	1/10 (10)	0/10	0/10
		ENR	3/10 (30)	1/10 (10)	2/10 (20)	1/10 (10)
	Overall	DIR	1/59 (1.7)	1/59 (1.7)	0/59	0/60
		ENR	4/59 (6.8)	1/59 (1.7)	2/59 (3.4)	2/60 (3.3)
Salmonella	132	DIR	0/18	0/18	0/18	0/18
		ENR	6/18 (33)a	2/18 (11)a,b	5/18 (28)a	0/18b
	160	DIR	0/31	1/31 (3.2)	0/31	0/32
		ENR	0/31	3/31 (10)	2/31 (6.5)	1/32 (3.1)
	181	DIR	0/10	0/10	0/10	0/10
		ENR	0/10	0/10	0/10	0/10
	Overall	DIR	0/59	1/59 (1.7)	0/59	0/60
		ENR	6/59 (10.2)	5/59 (8.5)	2/59 (3.4)	6/60 (10)

Fecal samples were collected on three occasions during the feeding period and cultured quantitatively (DIR) and qualitatively (ENR).

a,b

Row values (percentages) with different superscripts differ (p < 0.05).

DOF, days on feed; WDG, wet distiller's grains; SFC, steam-flaked corn; DRC, dry-rolled corn.

The majority (87.5%) of putative FC isolates examined were susceptible to all of the antibiotics on the National Antibiotic Resistance Monitoring System's panel (Table 2). Of those isolates with demonstrated resistance to one or more antimicrobials, 3 and 4 isolates were from the SFC and DRC treatments, respectively, and one isolate was from each of the SFC + WDG and DRC + WDG treatments. All multidrug resistance (2, 3, or 4 antibiotics) was observed in isolates cultured from the DRC and SFC treatments (2 isolates in each treatment). Resistance was observed to a total of six different antibiotics (streptomycin, tetracycline, sulfisoxazole, nalidixic acid, kanamycin, and trimethoprim/sulfamethoxazole) (Table 2).

Table 2.

Antimicrobial Resistance and Patterns of Resistance of Generic Escherichia coli Isolates Cultured from Feedlot Cattle Fed Diets Containing Either Steam-Flaked Corn or Dry-Rolled Corn With and Without 20% Wet Distiller's Grains

Item	SFC	SFC + WDG	DRC	DRC + WDG
Number of isolates examined	18	18	18	18
Number of isolates pan susceptible	15	17	14	17
Number of isolates with resistance to one or more antimicrobial	3	1	4	1
Number of isolates resistant to:
1 antibiotic	1	1	2	1
2 antibiotics	1	0	0	0
3 antibiotics	0	0	2	0
4 antibiotics	1	0	0	0
Patterns of resistance:
Streptomycin (S)	1
Sulfisoxazole (Su)				1
Tetracycline (T)		1	2
S + T	1
Nalidixic acid + Trimethoprim/Sulfamethoxazole + Su + T	1
Kanamycin + S + Su			2

Discussion

Previous research examining the effect of feeding DG on fecal prevalence of E. coli O157:H7 and Salmonella has yielded conflicting results. Two studies examining the influence of DG in naturally infected feedlot cattle (Jacob et al., 2008a) and experimentally infected Holstein calves (Jacob et al., 2008c) both reported an increased fecal prevalence of E. coli O157:H7 when DG was included in the diet. Salmonella was not examined in these first two studies. In two subsequent experiments by the same authors, results from one study indicated that DG may have influenced E. coli O157:H7 prevalence, but interpretation was confounded by a sampling day interaction at the end of the feeding period (Jacob et al., 2008b). The last experiment failed to show any effect of WDG on fecal prevalence of E. coli O157:H7 (Jacob et al., 2009), and in both reports, no effect of DG on Salmonella was observed. Similar to the aforementioned study (Jacob et al., 2009), we did not see any influence of DG on fecal prevalence of E. coli O157:H7. It is likely that the low overall prevalence we observed (4.2%) and observed by Jacob et al. (2009) (5.1%) was insufficient to detect treatment differences in naturally infected cattle. Although in one study (Jacob et al., 2008a) where significant DG effects were observed in naturally infected cattle, the mean prevalence was only 7.1%.

Contrary to previous research (Jacob et al., 2008b, 2009) reporting no effect of DG on Salmonella, we did see an effect of DG on fecal Salmonella prevalence. Feeding DRC + DG decreased the prevalence of Salmonella in cattle 132 DOF (28% vs. 18%) compared with those fed DRC only, and produced a similar numerical decrease in cattle fed SFC + DG (11%) compared with those fed SFC only (33%). Reasons for these differences between studies or for the effect on Salmonella observed in the current study are unknown. Both experiments reporting no effect on Salmonella prevalence fed dried DG, whereas wet DG was fed in the current research. Possibly, this affected a DG compound(s) that exerts an effect on Salmonella with the gastrointestinal tract of cattle. Others have hypothesized that DG may influence fecal prevalence of E. coli O157:H7 as a result of direct stimulation of this bacteria by a component of DG or via an indirect effect of DG on gut microbial populations (Jacob et al., 2008a). Although results of feeding DG on Salmonella prevalence in the current study cannot rule out the former, they do tend to support the latter hypothesis. Inclusion of DG significantly decreased Salmonella prevalence after 132 DOF when fed with DRC, whereas no difference was observed when DG was fed with SFC. The starch portion of corn is largely consumed in the ethanol production process, leaving a DG by-product that is high in fiber and protein, which when fed at 20% of the diet, would be expected to produce a different gastrointestinal environment. Steam-flaking of grains enhances ruminal digestion of the starch component within the rumen and may explain the difference in the current study due to feeding SFC or DRC with DG. If the influence was simply a matter of a compound within the DG, then we would have expected to see a similar inhibitory effect on Salmonella regardless of corn type fed in the current research. Granted, this is speculation based on a limited number of positive samples from one feedlot experiment, and previously published unexplained fluctuations of Salmonella prevalence are expected for naturally infected animals (Edrington et al., 2009).

Antimicrobials, including penicillin G, streptomycin, tetracycline, monensin, and virginiamycin, are used in the ethanol production process to suppress bacterial growth and increase yield (Narendranath et al., 2000). To our knowledge there is not any data to support the idea that antibiotics or antibiotic residues are escaping destruction in the distillation process and contained in the DG by-products. Nevertheless, genetic elements of resistance could theoretically be present in the coproduct. Wet DG, as fed in the current research, is reported to have a significant population of lactobacilli (Pederson et al., 2004). Taken together, this suggests that if genetic elements of resistance did make it through the distillation process, they would have had a host in the end product (DG) providing a source of antibiotic-resistant genes. Results of susceptibility testing of FC isolates cultured in the current study, however, found very little antimicrobial resistance overall, and even less in those isolated from the treatments feeding DG. As resistance patterns in the isolates from the WDG treatments (tetracycline and sulfisoxazole) were also observed in the corn-only treatments, they cannot be attributed to inclusion of WDG. Others have similarly concluded that feeding WDG was not associated with antimicrobial resistance (Jacob et al., 2008b).

Results of the current research found no significant effect of WDG on fecal prevalence of E. coli O157:H7 and only modest differences (decreases) in Salmonella prevalence. Additionally, there was no apparent effect on antimicrobial susceptibility of generic E. coli isolates associated with feeding of WDG.

Disclaimer

Mention of trade names, proprietary products, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

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.

Edrington

, Carter

, Friend

, Hagevoort

, Poole

, Callaway

, Anderson

, Nisbet

. Influence of sprinklers, used to alleviate heat stress, on faecal shedding of E. coli O157:H7 and Salmonella and antimicrobial susceptibility of Salmonella and Enterococcus in lactating dairy cattle. Lett Appl Microbiol, 2009; 48:738–743.

Fox

, Depembusch

, Drouillard

, Nagaraja

. Dry-rolled or steam-flaked grain-based diets and feral shedding of Escherichia coli O157 in feedlot cattle. J Anim Sci, 2007; 85:1207–1212.

Fron

, Madeira

, Richards

, Morrison

. The impact of feeding condensed distillers byproducts on rumen microbiology and metabolism. Anim Feed Sci Technol, 1996; 61:235–245.

Jacob

, Fox

, Drouillard

, Renter

, Nagaraja

. Effects of dried distiller's grain of fecal prevalence and growth of Escherichia coli O157 in batch culture fermentations from cattle. Appl Environ Microbiol, 2008a. 74:38–43.

Jacob

, Fox

, Drouillard

, Renter

, Nagaraja

. Evaluation of feeding dried distiller's grains with soluble and dry-rolled corn on the fecal prevalence of Escherichia coli O157:H7 and Salmonella spp. in cattle. Foodborne Pathog Dis, 2009; 6:145–153.

Jacob

, Fox

, Narayanan

, Drouillard

, Renter

, Nagaraja

. Effects of feeding wet corn distiller's grains with soluble with or without monensin and tylosin on the prevalence and antimicrobial susceptibilities of fecal foodborne pathogenic and commensal bacteria in feedlot cattle. J Anim Sci, 2008b. 86:1182–1190.

Jacob

, Parsons

, Shelor

, Fox

, Drouillard

, Thomson

, Renter

, Nagaraja

. Feeding supplemental dried distiller's grains increases faecal shedding of Escherichia coli O157 in experimentally inoculated calves. Zoonoses Public Health, 2008c. 55:125–132.

Narendranath

, Thomas

, Ingledew

. Urea hydrogen peroxide reduces the numbers of Lactobacilli, nourishes yeast, and leaves no residues in the ethanol fermentation. Appl Environ Microbiol, 2000; 66:4187–4192.

10.

National Agricultural Statistics Service. Ethanol co-products used for livestock feed. 2007. www.nass.usda.gov. 2009 March 23(Online.)

11.

Pederson

, Jonsson

, Lindberg

, Ross

. Microbiological characterization of wet wheat distiller's grain, with focus on isolation of lactobacilli with potential as probiotics. Appl Environ Microbiol, 2004; 70:1522–1527.

12.

Robinson

, Wright

, Williams

, Hart

, French

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