Salmonella and other Enterobacteriaceae in dairy-cow feed ingredients: antimicrobial resistance in western Oregon. (Features).

Introduction

Members of the Enterobacteriaceae family are becoming a source of increased health concern in food safety and clinical medicine (Brock & Madigan, 1991; Wall et al., 1994). In 1996, estimates of medical and lost-productivity costs associated with Salmonella species and Escherichia coli O157:H7 ranged from 0.2 to 3.5 billion dollars (Buzby, Roberts, Lin, & MacDonald, 1996). During 1990-1992, the genera Escherichia, Klebsiella, Enterobacter, Serratia, and Citrobacter were responsible for almost one-third of the nosocomial infections in the United States. The development of antibiotic-resistant strains of these organisms, with subsequent introduction into a health care setting by, for example, an infective patient, might substantially increase the health risks from nosocomial infections. In 1992, patients with nosocomial infections stayed an average of four additional days in the hospital and increased the cost of medical care by 4.5 billion dollars (Guentzel, 1999).

Several studies have suggested an association between the use of antimicrobial agents in animal feeds and an increased risk that humans will contract infections by resistant bacterial strains such as Salmonella spp., E. coli, and other enteric isolates (Al-Sam, Linton, Bennett, & Hinton, 1993; Centers for Disease Control and Prevention [CDC], 1997; Fontaine et al., 1978; Fraser & Mays, 1986; Glynn et al., 1998; Harris, Fedorka-Cray, Gray, Thomas, & Ferris, 1997; Hinton & Linton, 1986; Hinton, Hampson, & Linton, 1985; Holmberg, Wells, & Cohen, 1984; International Food Science Technologists, 1997; Jukes, 1955; Lee, Puhur, Maloney, Bean, & Tauxe, 1994; MacDonald et al., 1987; Mercer, Pocurull, Gaines, Wilson, & Bennett, 1971; Parry, Salmon, Willshaw, & Cheasty, 1998; Ramos, Ales, Cuenca-Estrella, Fernandez-Roblas, & Soriano, 1996; Siegal, Huber, & Enloe, 1974; Threnlfal, Frost, Ward, & Rowe, 1996; Threnlfal, Rowe, & Ward, 1993). Other studies have isolated various types of Salmonella spp. from animal feeds and o ther food products (Chang, 1975; Fone & Barker, 1994; Holmberg, Osterholm, Senger, & Cohen, 1984; Jones, 1971; Lyons et al., 1980; Skovgaard & Nielsen, 1972; Veldman, Vahl, Borggreve, & Fuller, 1995; Wall et al., 1995; Williams, Rollins, Pocurull, Selwyn, & Mercer, 1978).

A recent study by the Union of Concerned Scientists (Mellon, Benbrook, & Benbrook, 2001) reported that approximately 70 percent of the 25 million pounds of antibiotics used in the United States each year is fed to chickens, pigs, and cows for nontherapeutic reasons such as growth enhancement. Margaret Mellon, one of the report's authors, writes, "The government should act now to collect needed data [to design effective programs to prevent and control antibiotic-resistant strains of bacteria]" and adds, "The price of complacency could set us back to an era where untreatable infectious diseases are regrettably commonplace."

The objective of the current study was to evaluate whether animal feeds might serve as sources of antimicrobial-resistant bacteria, especially bacteria that are pathogenic to humans. To explore this hypothesis, the following study objectives were pursued:

1. identify Salmonella spp. and other Enterobacteriaceae in dairy-cow-feed ingredient piles on farms and test their patterns of antimicrobial resistance;

2. determine the prevalence of Salmonella spp. in the feed piles; and

3. determine whether the prevalence of Salmonella increases in individual piles as the piles age.

Methods

Study Population

The authors contacted 32 commodity-feeding dairy farms (defined as dairy farms feeding individual grain and crop by-products that are stored and mixed on the farm rather than premixed at a commercial feed mill, delivered to the farm, and fed as a single product). The farms were selected at random from a list of all 43 commodity-feeding dairy farms located in the mid-Willamette Valley of western Oregon. Of the 32 selected, 16 could not be contacted after repeated attempts. Of the farmers contacted, 12 agreed to participate in the study, two indicated that their farms no longer were commodity-feeding dairy farms, and two declined to participate, citing the busyness of the season.

Feed Ingredient Samples

From July through August 1998, samples of feed ingredients were collected from each of the 12 participating farms (a minimum of 30 to 35 ingredient piles was the desired sample size). The authors collected samples using sterile latex gloves and sterile spoons and placed the samples in Whirlpak sterile plastic bags (Nasco, Fort Atkinson, Wisconsin). Each bag contained between 100 and 200 grams of feed ingredients. The feed ingredients then were stored at 4[degrees]C to 8[degrees]C during transportation and refrigerated (at 2[degrees]C to 8[degrees]C) until the day of testing.

Two sampling strategies were employed: a prevalence survey and a repeated-samples survey, as described in the following subsections.

Prevalence Survey

For the prevalence survey, all of the feed ingredient piles available at the time of sampling were included in the study Typically the piles of different feed ingredients were located near each other, although they were always separated by at least one wall. The prevalence survey consisted of taking a onetime set of four individual samples from each ingredient pile. The authors sampled both newly acquired piles (those that had arrived at a farm within one week of sampling) and aged piles (those that had arrived at a farm more than one week before sampling). The samples were analyzed as described below in the section titled Laboratory Procedures.

To obtain a representative sample of feed ingredients, samples were taken from the left side, the right side, the center, and the subsurface of each pile. The left- and right-side samples were taken approximately 1 foot from the proximate wall of each feed ingredient bin. For all of the subsurface samples, except for that taken from the whole-cottonseed pile, a tube-shaped, sanitized trier was inserted (three to four times) into the front of each pile to collect the sample. The trier was approximately 3 feet long and had a hole 1 inch in diameter. Both the trier and the rubber boots worn by the sampler were sanitized with a 15 percent bleach/water solution and then rinsed with clean tap water after each sample was taken to prevent cross-contamination.

Because the cotton residue on the cottonseeds deterred subsurface sampling with the trier, a 1-foot-by-1-foot hole was dug into the whole-cottonseed pile to obtain subsurface sampling, which was done with a sterile spoon.

Repeated-Samples Survey

The repeated-samples survey consisted of sampling a subset of piles over time. All feed ingredient piles that were less than one week old at initial sampling were enrolled in the repeated-samples survey. Using sterile latex gloves and sterile spoons, the authors took two surface samples, from the left and right sides of each pile, approximately 1 foot from the walls of the food bin. Each feed ingredient pile was sampled weekly until the pile was depleted.

Laboratory Procedures

All samples (from both the prevalence survey and the repeated-samples survey) were tested individually. A 25-gram sample of each feed ingredient was placed into 225 milliliters (mL) of buffered peptone water (BPW) and stirred with a sterile spoon. After an incubation period of 18 to 24 hours (at 35[degrees]C [+ or -] 0.5[degrees]C), 0.1 mL of BPW was transferred to 10 mL of Rappaport-Vassiliadis (RV) broth, and 1 mL of BPW was transferred to 10 mL of tetrathionate (TT) broth (DIFCO, Detroit, Michigan). To each container of TT broth was added 0.1 mL of 0.1 percent brilliant green (DIFCO) and 0.2 mL of iodine solution. After the RV and TT broths were incubated for six to eight hours at 42[degrees]C [+ or -] 0.5[degrees]C, 0.5 mL of the RV broth and 0.5 mL of the TT broth were transferred to 9 mL of M-Broth (DIFCO). The M-Broth was incubated for 18 to 24 hours at 42[degrees]C [+ or -] 0.5[degrees]C (Andrews, June, Sherrod, Hammack, & Amaguana, 1995; U.S. Department of Agriculture [USDA], 1997).

The authors tested the M-Broth for the presence of Salmonella spp. using Assurance Salmonella EIA (an ELISA, or enzyme-linked immunoabsorbent assay test, obtained from BioControl Systems, Inc., of Bothell, Washington, and performed by an Immunotech automated ELISA machine from Italy). As control strains, Salmonella luciana and Citrobacter species were obtained from the bacterial-culture collection of the Oregon Department of Agriculture (ODA) Laboratory Services Division. American Type Culture Collection (ATCC) strains Escherichia coli (ATCC 11775), Klebsiella pneumoniae (ATCC 13883), and Enterobacter aerogenes (ATCC 13048) also served as control organisms (Feldsine, Falbo-Nelson, & Hustead, 1993; Andrews et al., 1995).

M-Broths that were presumptive positive for Salmonella were streaked onto a xylose lysine agar base with tergitol 4 supplement (XLT4) (DIFCO) and onto brilliant-green medium with novobiocin antimicrobic supplement (BGN plates) (DIFCO). The XLT4 and BGN plates were incubated for 18 to 24 hours at 35[degrees]C [+ or -] 0.5[degrees]C. Colonies that grew on the plates were streaked and stabbed onto triple sugar iron (TSI) (DIFCO) and lysine iron agar (LIA) (BBL, Cockeysville, Maryland) slants, The TSI and LIA slants were incubated for 18 to 24 hours at 35[degrees]C [+ or -] 0.5[degrees]C (Andrews et al., 1995; Brock & Madigan, 1991; DIFCO, 1977; USDA, 1997).

Oxidase-negative (Oxidase Reagent Spot Test, DIFCO) isolates from the TSI and LIA slants were biochemically identified with the Enterobacteriaceae Microbiological Identification (Micro-ID) kit system (Remel, Lenexa, Kansas). If the Enterobacteriaceae Micro-ID system identified an isolate as Salmonella, the isolate was serogrouped with the somatic (O) group agglutination test and serotyped with the Spicer-Edwards flagellar (H) agglutination test (Bacto-Salmonella Antisera, DIFCO). The motility of any Salmonella spp. bacteria was tested with the GI motility medium (Andrews et al., 1995; DIFCO, 1977).

In vitro antibiotic-sensitivity testing of all identified isolates was determined with an agar disk diffusion method. Zone interpretations and laboratory protocols were based on the recommendations of the National Committee on Clinical Laboratory Standards (NCCLS). Antimicrobial disks (Sensi-Disc, BBL, Cockeysville, Maryland) included ampicillin (10 micrograms [[micro]g]), chloramphenicol (30 [micro]g), ciprofloxacin (5 [micro]g), streptomycin (10 [micro]g) and tetracycline (30 [micro]g). Bacterial controls (QualiSwab, BBL) included Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), and Enterococcus faecalis (ATCC 29212). A trimethoprim disk (Sensi-Disc, BBL) was used to detect high levels of thymine and thymidine in the Mueller Hinton medium, which could have distorted results (Barry & Thornsberry, 1991; Bauer et al., 1966; NCCLS, 1995).

The agar disk diffusion method (based on the Kirby-Bauer method) uses three zone-diameter categories (in millimeters) to characterize the antimicrobial resistance or sensitivity of a microorganism. The zone-diameter measurements categorize antimicrobial sensitivity as "resistant," "intermediate," or "susceptible." "Resistant" is the minimum zone diameter indicating that a microorganism will be resistant to an antimicrobial at normal human therapeutic doses. "Susceptible" is the minimum zone diameter indicating that a microorganism will be susceptible to the antimicrobial at normal therapeutic doses. "Intermediate" is a range of zone diameters between susceptible and resistant zone diameters for which individual differences among patients and strains may result in either a susceptible or a resistant outcome (Barry & Thornsberry, 1991; Bauer, Kirby, Sherris, & Turck, 1966; NCCLS, 1995).

Results

Prevalence Survey: Salmonella Results

Fifty feed ingredient piles were sampled for the prevalence survey. The feed ingredients included beet pulp (pelleted), grass seed screenings (pelleted), canola meal, rapeseed, soybean meal, canola (pelleted), wheat mill run, cottonseed (whole), corn and barley mix, rolled grain, soy pak, corn distillers' grains, corn gluten (pelleted), and corn (flaked).

Forty-two percent of the piles (21 of 50) were presumptive positive for Salmonella spp. (Assurance Salmonella EIA kit; Table 1). By the culture method and Entero-bacteriaceae Micro-ID system, 2,0 percent (one of 50) were confirmed as S. enteritidis and serogrouped as poly Group B, Group [C.sub.1]. Although the S. enteritidis isolate was motile, its flagellar (H) antigens could not be determined. The S. enteritidis isolate was recovered from a whole-cottonseed pile at Farm 1.

Repeated-Samples Survey: Salmonella Results

Ten feed ingredient piles from three different farms were sampled during the repeated-samples survey (Table 2). Feed ingredients included beet pulp (pelleted), grass seed screenings (pelleted), canola meal, rapeseed, cottonseed (whole), corn distillers' grains, and soybean meal. Overall, 60.0 percent of the piles (six of 10) were presumptive positive for Salmonella spp. By the culture method and the Enterobacteriaceae Micro-ID system, 20.0 percent of the piles (two of 10) were confirmed positive for S. enteritidis and serogrouped as poly Group B, Group [C.sub.1]. The S. enteritidis isolates were recovered from Farm 1, in a soybean meal pile (Week 2) and in a whole-cottonseed pile (Week 4). Their flagellar (H) antigens could not be determined. At Farm 1, all three sampled piles (soybean meal, corn distillers' grains, and cottonseed piles) were presumptive positive for Salmonella spp. as the piles aged. At Farm 12, three feed piles remained negative for Salmonella spp. throughout the repeated-samples study Thre e of the four piles at Farm 5, either initially or over time, were presumptive positive for Salmonella spp. (Table 2).

Analysis of Overall Antimicrobial Resistance

The Enterobacteriaceae Micro-ID system identified 50 bacterial isolates from the prevalence and repeated-samples surveys (42 isolates from the prevalence survey and eight isolates from the repeated-samples survey). All 50 isolates were tested for resistance to ampicillin, chloramphenicol, ciprofloxacin, streptomycin, and tetracycline.

As shown in Table 3 (results from the prevalence survey) and in Table 4 (results from the repeated-samples survey), all Enterobacteriaceae isolates tested were fully resistant to ampicillin, tetracycline, streptomycin, or a combination of the three. Overall, 62.0 percent of the Entero-bacteriaceae isolates (31 of 50) showed ampicillin resistance. All of the Enterobacter cloacae, Enterobacter gergoviae, Enterobacter agglomerans, Enterobacter aerogenes, Citrobacter diversus, Klebsiella pneumoniae, and Serratia liquefaciens isolates were ampicillin resistant. Half of the Hafnia alvei isolates (three of six) were resistant to ampicillin. Of the Citrobacter species, the one C. diversus isolate and one-third of the C. freundii isolates (two of six) manifested ampicillin resistance. One of the nine E. coli isolates was resistant to ampicillin.

Overall, 10.0 percent of the Enterobacteriaceae isolates (five of 50) had tetracycline resistance, Four of the nine E. coli isolates showed tetracycline resistance, and one of the six K. pneumoniae isolates had tetracycline resistance.

Other members of the Enterobacteriaceae family displayed intermediate resistance to various antimicrobials (Table 3 and Table 4). Overall, 14 percent of the Enterobacteriaceae isolates (seven of 50) manifested intermediate resistance to ampicillin. Approximately 11.0 percent of the E. coli isolates (one of nine) showed intermediate resistance to tetracycline, and one-third of the E. coli isolates (three of nine) showed intermediate resistance to ampicillin. Two of the nine E. coli isolates had intermediate resistance to chloramphenicol, and two had intermediate resistance to streptomycin. Twenty-five percent of the S. enteritidis isolates (one of four) showed intermediate resistance to ampicillin. Of the K. pneumoniae isolates, 16.7 percent (one of six) had intermediate resistance to streptomycin while half of the C. freundii isolates (three of six) had intermediate resistance to ampicillin. Half of the S. Iiquefaciens isolates (two of four) had intermediate resistance to chloramphenicol. The C. diversus isol ate that was resistant to ampicillin also showed intermediate resistance to tetracycline.

One of the nine E. coli isolates and one of the six K. pneumoniae isolates were resistant to multiple drugs (Table 3 and Table 4). The E. coli isolate showed resistance to streptomycin and tetracycline while the K. pneumoniae isolate showed resistance to both ampicillin and tetracycline.

None of the bacterial isolates exhibited any resistance or intermediate resistance to ciprofloxacin.

Discussion

Only a few studies have evaluated the extent to which antimicrobial-resistant microbes contaminate the feeds of human-food-producing animals (Durand, Barnard, Swanepoel, & Engelbrecht, 1987; Harris et al., 1997; Schwalbe, McIntosh, Qaiyumi, Johnson, & Morris, 1999). A previous study has, for example, isolated antimicrobialresistant Salmonella spp. and E. coli from fecal samples of farm animals (Mamber & Katz, 1985). In another study serotype S. typhimurium, phage type 44, isolated from fecal samples, showed 100 percent resistance to ampicillin, chloramphenicol (banned, in the United States and Canada, from use in food-producing animals), streptomycin, and tetracycline (Mackie, Lightfoot, Adamson, & Wishart, 1996). In another study 15 to 40 percent of E. coli isolates from farm animals showed resistance to ampicillin (Jukes, 1955). The high frequency of ampicillinresistant Enterobacteriaceae and the frequency of resistance to streptomycin and tetracycline observed in the current study mirror the high prevalenc e of ampicillin-resistant strains, and the presence of streptomycinand tetracycline-resistant strains, observed in previous studies.

Antibiotics used on the dairy farms participating in this study include ampicillin (represented by the other betalactams, penicillin, cloxicillin, and amoxicillin); streptomycin; gentacin; and tetracyclines. Ciprofloxacin is not used. Most of the antibiotics are infused in the udder, injected into the muscle or under the skin, or given intravenously as therapy for bacterial infections or prevention against such infections. Chlorotetracycline is included in calf milk replacer and calf starter grain as a growth promoter. It may be fed in grain only during the spring and fall, not year-round.

The tetracycline disc used in the current study is a class disc for all tetracyclines that are used to treat animal and human diseases. It has zone diameters that indicate resistance to chlortetracycline, oxytetracycline, demeclocycline, doxycycline, methacycline, and minocycline. The ampicillin disc used in this study is the class disc for penicillins such as ampicillin, amoxicillin, and hetachillin. In addition, the ampicillin disc is a class disc for bacampicillin and cyclacillin. Thus, the study results indicate significant resistance to a broad range of antimicrobials used to treat human disease (Barry & Thornsberry 1991; Bauer et al., 1966, NCCLS, 1995).

Because many of the Enterobacteriaceae isolated in this study are enteric microorganisms, fecal contamination of the feed ingredients seems likely. Such contamination may have occurred during the transport of feed ingredients to the storage bin, a process that typically is accomplished through the use of bulldozers. On the other hand, because the storage feed bins are not protected from animals that can carry Salmonella spp. and other enterics in their feces (for example, dairy calves, rodents, and birds), contamination also could have occurred through this route (Blood & Radostits, 1989; Brock & Madigan, 1991). It would have been interesting to culture feces from these animals on the study farms.

Because the paths of bacterial contamination are complex and varied, other sources of contamination are possible. For example, bacterial contamination of feed ingredients could occur before the ingredients arrive on a farm, either at a feed mill, from transportation vehicles, or during feed storage (CDC, 1997; Muirhead, 1995; NCCLS, 1995). Feed ingredients such as canola meal and whole cottonseed often are waste products of other processing operations. Thus, contamination of these feed ingredients may have occurred before or during the recycling process. Farm-specific factors also might affect the prevalence of bacterial contamination; although whole-cottonseed and soybean-meal piles were obtained and tested from several farms, S. enteritidis was recovered only from samples taken at Farm 1. This finding raises the question of whether the isolate originated from an environmental source or from some other source unique to Farm 1 (Blood & Radostits, 1989; Ensminger & Olentine, 1978).

The presence of antimicrobial-resistant Enterobacteriaceae in dairy-cow feed ingredients raises serious questions about whether those microorganisms might enter the human food supply and infect people. In addition, the microorganisms may pose health risks to farmers and farm visitors (samples from those groups were not cultured in this study), especially if they practice poor handwashing techniques or are immunocompromised (Holmberg, Osterholm, et al., 1984; Johnston, Bruce, & Hill, 1983; Lyons et al., 1980).

Conclusion

This study raises several unanswered questions regarding the high prevalence of antimicrobial-resistant bacterial strains found in dairy cow feeds. Through what routes are the feed ingredient piles becoming contaminated with antimicrobial-resistant bacteria that are pathogenic to humans? In particular; to what extent, if any, is the widespread use of antimicrobials on dairy farms and on other food-producing farms contributing to the eventual loss of antimicrobials as successful treatments for human bacterial infections? What human health effects, now or in the future, can or will be attributable to ingestion of antimicrobial-resistant human pathogens by food-producing animals? Also, if feed piles are shown to be a significant source of antimicrobial-resistant infections in humans, what contamination prevention and control strategies must be implemented?

Although the answers to those questions are still to be found, antimicrobial-resistant contamination in feed ingredients was observed in this study to occur on a magnitude that warrants careful consideration of such contamination as an emerging and potentially serious environmental health problem.

TABLE 1

Prevalence Survey: Salmonella Presumptive Positives and Feed Types

Feed Type                         Number of      Number of
                                    Piles    Piles Presumptive
                                                 Positive

Beet pulp (pelleted)                  2              1
Canola (pelleted)                     1              0
Canola meal                           9              6
Corn (flaked)                         2              1
Corn/barley mix                       5              2
Corn distillers' grains               7              2
Corn gluten (pelleted)                2              0
Cottonseed (whole)                   10              5
Grass seed screenings (pelleted)      1              1
Rapeseed                              1              0
Rolled grain                          1              1
Soybean meal                          6              0
Soy pak                               1              0
Wheat mill run                        2              2

Total                                50             21

Feed Type                         Percentage



Beet pulp (pelleted)                 50.0
Canola (pelleted)                     0.0
Canola meal                          66.7
Corn (flaked)                        50.0
Corn/barley mix                      40.0
Corn distillers' grains              28.6
Corn gluten (pelleted)                0.0
Cottonseed (whole)                   50.0
Grass seed screenings (pelleted)    100.0
Rapeseed                              0.0
Rolled grain                        100.0
Soybean meal                          0.0
Soy pak                               0.0
Wheat mill run                      100.0

Total                                42.0
TABLE 2

Repeated-Samples Survey: Salmonella Presumptive Positives and Feed Types

Feed Type                         Farm   Number     Week of
                                  Code  of Weeks  Presumptive
                                        Sampled    Positive

Corn distillers' grains            1       3           2
Cottonseed (whole)                 1       4         2, 4
Soybean meal                       1       3         2, 3
Beet pulp (pelleted)               5       5        1, 2, 5
Grass seed screenings (pelleted)   5       5         1, 3
Canola meal                        5       4           1
Rapeseed                           5       5           0
Beet pulp (pelleted)               12      6           0
Canola meal                        12      3           0
Corn distillers' grains            12      4           0

Feed Type                          Number of
                                  Presumptive
                                   Positives

Corn distillers' grains                1
Cottonseed (whole)                     2
Soybean meal                           2
Beet pulp (pelleted)                   3
Grass seed screenings (pelleted)       2
Canola meal                            1
Rapeseed                               0
Beet pulp (pelleted)                   0
Canola meal                            0
Corn distillers' grains                0
Table 3

Antimicrobial-Resistance Patterns of Enterobacteriaceae Isolates from
the Prevalence Survey (*)

Isolate             Feed                     A  CH  C  ST  T

S. enteritidis      Cottonseed               I  S   S  S   S
S. enteritidis      Cottonseed               S  S   S  S   S
E. aerogenes        Canola meal              R  S   S  S   S
E. agglomerans      Canola meal              R  S   S  S   S
E. cloacae (**)     Cottonseed               R  S   S  S   S
E. cloacae          Cottonseed               R  S   S  S   S
E. cloacae          Cottonseed               R  S   S  S   S
E. cloacae          Rolled grain             R  S   S  S   S
E. cloacae          Corn distillers' grains  R  S   S  S   S
E. cloacae (**)     Canola meal              R  S   S  S   S
E. cloacae          Canola meal              R  S   S  S   S
E. cloacae          Canola meal              R  S   S  S   S
E. cloacae          Beet pulp                R  S   S  S   S
E. cloacae          Canola meal              R  S   S  S   S
E. gergoviae        Canola meal              R  S   S  S   S
H. alvei            Corn/barley mix          R  S   S  S   S
H. alvei            Canola meal              S  S   S  S   S
H. alvei            Canola meal              R  S   S  S   S
H. alvei            Corn/barley mix          S  S   S  S   S
H. alvei            Cottonseed               R  S   S  S   S
H. alvei            Canola meal              S  S   S  S   S
E. coli             Soybean meal             S  S   S  I   R
E. coli             Wheat mill run           R  S   S  S   S
E. coli (**)        Corn/barley mix          S  S   S  S   S
E. coli             Cottonseed               S  S   S  S   I
E. coli             Cottonseed               I  I   S  S   R
E. coli             Cottonseed               I  I   S  I   R
E. coli             Grass seed               S  S   S  S   S
C. freundii         Wheat mill run           R  S   S  S   S
C. freundii         Wheat mill run           I  S   S  S   S
C. freundii         Wheat mill run           R  S   S  S   S
C. freundii         Cottonseed               I  S   S  S   S
C. freundii         Corn/barley mix          I  S   S  S   S
K. pneumoniae       Wheat mill run           R  S   S  I   R
K. pneumoniae (**)  Canola meal              R  S   S  S   S
K. pneumoniae       Corn/barley mix          R  S   S  S   S
K. pneumoniae (**)  Beet pulp                R  S   S  S   S
K. pneumoniae       Canola meal              R  S   S  S   S
S. liquefaciens     Cottonseed               R  S   S  S   S
S. liquefaciens     Flaked corn              R  I   S  S   S
S. liquefaciens     Flaked corn              R  S   S  S   S
S. liquefaciens     Corn distillers' grains  R  I   S  S   S

(*)Samples from the first week of the repeated-samples survey are
included in the table.

(**)Sample from the first week of the repeated-samples survey.

A = ampicillin.

C = ciprofloxacin.

CH = chloramphenicol.

I = intermediate resistance.

R = resistance.

S = susceptible.

ST = streptomycin.

T = tetracycline.

Accuracy criteria: Mean of Niton readings must be within 95% to 105% of
the mean of the laboratory results (plus or minus 5%).
TABLE 4

Antimicrobial-Resistance Patterns of Enterobacteriaceae Isolates from
the Repeated-Samples Survey (*)

Isolate             Week of Isolation  A  CH  C  ST  T

S. enteritidis              4          S  S   S  S   S
S. enteritidis              2          S  S   S  S   S
E. cloacae (**)             1          R  S   S  S   S
E. cloacae (**)             1          R  S   S  S   S
E. aerogenes                4          R  S   S  S   S
E. coli                     2          S  S   S  R   R
E. coli (**)                1          S  S   S  S   S
E. coli                     3          I  S   S  S   S
C. freundii                 4          S  S   S  S   S
C. diversus                 4          R  S   S  S   S
K. pneumoniae               5          R  S   S  S   S
K. pneumoniae (**)          1          R  S   S  S   S
K. pneumoniae (**)          1          R  S   S  S   S

(*)Samples from Week 1 also are included in the prevalence survey.

(**)Sample included in the prevalence survey.

A = ampicillin.

C = ciprofloxacin.

CH = chloramphenicol.

I = intermediate resistance.

R = resistance.

S = susceptible.

ST = streptomycin.

T = tetracycline.

Acknowledgement: The authors gratefully acknowledge the generosity of the Oregon Department of Agriculture Laboratory Services Division for providing laboratory workspace and supplies for microbial analyses.

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Corresponding Author: Annette M. Rossignol, Sc.D., Professor, College of Health and Human Performance, Oregon State University, Corvallis, OR 97331-6406. E-mail: <[e-mail]>.