Novobiocin

Abstract

Escherichia coli O157:H7 has been a major cause of human illness outbreaks in the U.S., mainly attributable to the consumption of contaminated beef. Worldwide, however, other shiga toxin-producing E, coli (STEC) have been responsible for major outbreaks of human illnesses. Because of the lack of sorbitol fermentation and [beta]-glucuronidase activity, E. coli O157:H7 is relatively easier to detect than other STEC. The objective of this experiment was to examine prevalence of sorbitol-negative STEC in beef heifers grazing rangeland forages over the four seasons of 1999. However, because of the loss of heifers and vegetation to wild fires during the summer, the study was terminated, and the results of two seasons (winter and spring) were reported. A random number of heifers (22 in winter; 23 in spring) from a herd of 70 were fecal-sampled; nine were sampled in both seasons. A random sample (n = 115) of potential sorbitol-negative STEC isolates was screened for the presence and expression of toxin genes. Fifty-six STEC isolates were detected in feces of five heifers in the winter and two heifers in the spring with occurrence rates of 22.7 and 8.7%, respectively. Of these isolates, 37 had and expressed Shiga Toxin 1(Stx1), 14 had and expressed Shiga Toxin 2 (Stx2), and five had but did not express stx2. Only non-O157:H7 STEC (O118:H^sup -^ [non-motile] and O138:H^sup -^) were detected. Because E. coli O118:H^sup -^ is pathogenic to humans, it is important to determine its incidence in cattle and to determine the risk of beef contamination with such a pathogen.

(Key Words: Escherichia coli, Beef Cattle, Foodborne Pathogens, Shiga Toxins, Food Safety.)

Introduction

For two decades (Riley et al., 1983), shiga toxin-producing Escherichia coll (STEC), including O157:H7, have been known to cause human illnesses (Fitzpatrick et al., 1991; Griffin and Tauxe, 1991) such as mild diarrhea, hemorrhagic colitis, and hemolytic uremie syndrome (HUS). The toxins produced (Bell et al., 1994) resemble those of Shigella dysenteriae (O'Brien and Holmes, 1987; Scotland and Smith, 1997). They are called Shiga Toxins 1 (Stxl) and 2 (Stx2) and are referred to as verotoxins (Konowalchuk et al., 1977). Although Stxl and Stx2 are different proteins encoded by different genes, their effects are similar (Acheson and Keusch, 1996). The STEC can produce one or both toxins (Scotland and Smith, 1997), and either one can cause human illnesses (Willshaw et al., 1997). The O157:H7 (Hussein et al., 1999) and non-O157:H7 (Bettelheim, 2003) STEC are more prevalent in cattle than other animals. Between 1982 and 1996, ground beef contaminated with E. coli O157:H7 was the cause of 40 human illness outbreaks in the U.S. (USDA-APHIS, 1997). As a result, many studies focused only on this serotype (Hancock et al., 1994; Faith et al., 1996; Rice et al., 1997). Worldwide, however, non-O157:H7 STEC were responsible for major outbreaks of human illnesses (Bettelheim, 2003). Testing in several countries (Beutin et al, 1993; Wilson et al., 1996; Blanco et al., 1997; Desmarchelier, 1997; Lopez et al., 1997; Miyao et al., 1998; Schurman et al., 2000; Zschock et al., 2000; Kobayash et al., 2001) also revealed that O157:H7 was only a small proportion of STEC in cattle feces.

In the past decade, non-O157:H7 STEC caused several U.S. outbreaks (CDC, 1995; Acheson and Keusch, 1996), and their threat to public health was emphasized (Tarr et al, 1996; Acheson et al., 1997). Our studies with heifers on irrigated pastures (Thran et al., 2001a,b) or cows on rangelands (Hussein et al., 2003a) revealed presence of O157:H7 and non-O157:H7 (O6:H^sup -^ [non-motile], O8:H^sup -^, O26:H^sup -^, O105:H^sup -^, and O141:H^sup -^) STEC that are known to cause human illnesses (Johnson et al., 1996; Bonnet et al., 1998; Strockbine et al., 1998; Bettelheim, 2003; Blanco et al., 2003). Because variations in occurrence and characterization of sorbitolnegative STEC existed among sheep grazing irrigated pastures or arid rangeland forages (Hussein et al., 2003b), this study examined prevalence of sorbitol-negative STEC in heifers grazing rangeland forages.

Materials and Methods

Animals and Sample Collection. A herd of 70 yearling Angus crossbred heifers at the University of Nevada Gund Research and Demonstration Ranch (Beowawe, NV) was used. During winter, the heifers grazed dormant crested wheatgrass (Agropyron desertomrri) and were supplemented with wheat middlings and alfalfa (Medicago sativa) hay. During spring, the heifers grazed crested wheatgrass and native sagebrush (Artemisia tridentata). This study was designed to assess occurrence of sorbitol-negative STEC over the four seasons of 1999. Because of the incidence of wild fires on the ranch during the summer of that year, however, we were forced to terminate the study and report only the results of the winter and spring. These wild fires caused the death of approximately 50% of the heifers used in this study and the burn of >80% of ranch forages. Therefore, it was impossible to replace the heifers because of the lack of grazing forages. The management of the heifers on the ranch was according to procedures approved by the Nevada Agricultural Experiment Station. The study was also conducted in accordance with the principles and specifie guidelines presented in Guidelines for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999). Forty-five fecal grab samples were directly removed from the rectum during two collection periods (winter [February] and spring [May]). These fecal samples were from heifers (22 in winter; 23 in spring) that were randomly selected from the herd at each sampling time. Nine heifers were sampled in both periods. Each fecal sample was placed in a sterile Whirl-pak bag (Nasco, Modesto, CA), and all samples were immediately returned to the laboratory for analysis.

Bacteriological Analyses. The initial selection of isolates to be screened for STEC status was conducted by adding 1.0 g of fresh feces to 25 mE of enrichment medium (tryptic soy broth [TSB] (Hardy Diagnostics, Santa Maria, CA) containing novobiocin (20 µg/mL; Sigma, St. Louis, MO) and vancomycin [40 µg/ mL; Sigma]). The feces and medium were mixed vigorously to suspend fecal material, and the suspension was incubated at 25°C for 20 min to allow for antibiotic selection. The optimal dilutions for plating were established by preliminary experiments. A portion of the fecal suspensions was serially diluted to 10^sup -2^, plated in duplicate onto sorbitol-MacConkey (SMAC) agar (Hardy Diagnostics) containing novobiocin (20 µg/mL) and vancomycin (40 µg/mL), and incubated at 37°C for 18 h. The fecal suspensions also were incubated at 37°C for 18 h with continuous shaking and then were serially diluted to 10^sup -7^. The 10^sup -5^, 10^sup -6^, and 10^sup -7^ dilutions were plated in duplicate onto SMAC agar containing novobiocin (20 µg/ mE) and vancomycin (40 µg/mE). The plates were incubated at 37°C for 18 h. all colonies were enumerated by using a Bantex Model 90OA illuminated colony counter (American Bantex Corp., Burlingame, CA).

Non-sorbitol fermenting bacteria (white colonies) on SMAC plates representing each heifer at each collection period were subcultured to 4-methylumbelliferyl-[beta]-D-glucuronide (MUG) MacConkey (MMUG; Hardy Diagnostics) agar grid plates. If a SMAC plate contained 10 or less sorbitol-negative (white) colonies, all were transferred to MMUG plates. However, if the SMAC plate had

Determination of Verotoxicity. The isolates (i.e., sorbitol-negative/ MUG-negative or sorbitol-negative/ MUG-positive) collected (n = 115) from the heifers were confirmed as E. coli by biochemical tests using the API 2OE identification system (bioMerieux Vitek, Inc., Hazelwood, MO). To prepare for verotoxicity testing, the E. coli isolates were grown in 5 mL of TSB at 37°C for 18 h, the cultures were centrifuged (3000 × g for 10 min), and the supernatants were filtered through 0.2-µm sterile syringe filters (ISC BioExpress, Kaysville, UT). The sterile supernatant was used to determine cytotoxicity via the use of Vero (African Green Monkey kidney) cells (Konowalchuk et al., 1977). Negative controls consisting of Eagle minimal essential medium (Mediatech, Inc., Herndon, VA), TSB medium, and non-STEC O157:H7 (ATCC 43888) were analyzed with each set of plates. In addition, each set of plates contained a positive control panel of supernatants from three E. coli O157:H7 isolates known to produce only Stxl (ATCC 43890), only Stx2 (ATCC 43889), or both toxins (ATCC 43895). The STEC-positive and STEC-negative isolates were determined by absence or presence of a confluent monolayer, respectively. Results were recorded after 24 and 48 h of incubation.

Detection of the Toxin Genes. The toxin genotype (stxi or stx2) of the 115 E. coli isolates was confirmed by polymerase chain reaction (PCR). In a single tube, a 50-µL PCR reaction mixture was prepared using MasterTaq kit (Eppendorf Scientific, Wesbury, NY) following the manufacturer's instructions (5 × TaqMaster PCR enhancer, 10 × Taq buffer with 0.15 rnM MgCl^sub 2^; 0.1 µL each 100 mM nucleotide; 1.0 unit Taq polymerase). A total of 100 ng of both forward and reverse primers were used for each reaction. Whole cells from each bacterial culture (1 µL liquid culture) were added to the PCR mixture instead of purified nucleic acids. The oligonucleotide primers (Pollard et al, 1990; McCleery and Rowe, 1995) were commercially prepared (Life Technologies GIBCO BRL, Rockville, MD). The PCR was performed using a Mastercycler Gradient thermocycler (Eppendorf Scientific) following the temperature program previously reported (Pollard et al., 1990). Amplified DNA fragments were visualized by gel electrophoresis on a 1% agarose containing ethidium bromide (10 mg/mL).

Sequencing of PCR Products. The PCR products of the correct size were excised and purified using the GeneClean kit (BiolOl Inc., La Jolla, CA) according to the manufacturer's instructions. The DNA was dried in a SpeedVac (Savant, Farmindale, NY) and was resuspended in sterile deionized water as needed for sequencing. For automatic sequencing, an Applied Biosystems DNA sequencer (ABI Prism 310 Genetic Analyzer; Perkin Elmer, Foster City, CA) and a dideoxy terminator cycle sequencing kit (Perkin Elmer) were used following the manufacturer's instructions. A 10-µL PCR reaction was set up according to the manufacturer's instructions. Briefly, a terminator reaction mix, purified PCR product (10 to 30 ng), and sequencing primer (15 ng) were used for this reaction. The PCR mixture was placed in a Mastercycler Gradient thermocycler (Eppendorf Scientific) programmed to 94°C for 10s, 5O°C for 5 s, and 60°C for 4 min for 25 cycles. Each sample was ethanol-precipitated (1 µL 3 M sodium acetate IpH 5.2], and 25 µL 100% ethanol) followed by drying. The pellet was resuspended in 15 µL of template suppression reagent (Perkin Elmer). The samples were denatured at 95°C for 2 min, quick-cooled on ice, transferred to sequencer tubes, and stored at -20°C until sequencing.

Expression of the Toxin Genes. The verotoxin-producing E. coli-reversed passive latex agglutination (VTEC-RPLA) assay (Denka Seiken Co., Ltd., Tokyo, Japan) was performed according to the manufacturer's instructions. Isolates were grown ' in 5 mL of TSB at 37°C for 18 h without shaking. From this culture, 1 mL was placed in a 1.5-mL eppendorf tube and centrifuged at 3000 × g for 20 min. In 96-well, V-bottom microtiter plates (Costar, Corning, NY), culture supernatants were mixed 1:1 (25 µL) with the supplied diluent. An equal volume of latex particles sensi- tized with rabbit polyclonal anti-Stxl or anti-Stx2 immunoglobulin G antibody was mixed in appropriate wells. Plates were covered, incubated at room temperature, and examined for latex agglutination after 18 h. The positive and negative control toxins supplied with the kit were run with each assay. A positive result was recorded when agglutination in the sample well was two levels above that of the control.

Determination of the O157:H7 Status. A colony blot was performed to establish the antigen profile (O157:H7 positive or negative) of the STEC isolates under a protocol modified from Hull et al. (1993). A 0.45-µm (85-mm) nitrocellulose membrane (Fisher, Atlanta, GA) was placed on top of a trypicase soy agar (Hardy, Santa Maria, CA) plate. The STEC isolates were subcultured in a grid pattern onto the membrane and incubated for 18 h at 37°C. The membrane was removed from the trypi case soy agar plate and blocked for 2 h with 5% nonfat milk in 10 mM Tris, 150 mM NaCl, and 0.05% Tween (pH 8). Following each step of the procedure, the membrane was washed with three 10-min washes of 10 mM Tris, 150 mM NaCl, and 0.05% Tween (pH 8). After blocking, the membrane was incubated with a 1:100 dilution of goat-anti-O157:H7 antibody (Kirkgaard and Perry Laboratories, Gaithersburg, MD) for 1 h, washed, and incubated for l h with a secondary antibody [alkaline phosphatase conjugated rabbit-anti-goat immunoglobulin G (Kirkgaard and Perry Laboratories)]. The membrane was developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (Promega, Madison, WI). The positive controls on the blot consisted of known O157:H7 E. coli cultures (ATCC 43895 and 43889), and the negative control was a non-O157:H7 E. coli (ATCC 25922).

Serotyping of the STEC Isolates. The STEC isolates were serotyped at the E. coli Reference Center (Pennsylvania State University, University Park).

Results and Discussion

Fifty-six sorbitol-negative STEC isolates (Table 1) were detected in feces of five heifers (i.e., 1, 2, 12, 18, and 4 isolates from Heifers A, B, C, D, and E, respectively) in the winter and from two other heifers (i.e., 6 and 13 isolates from Heifers F and G, respectively) in the spring. The STEC occurrence rates in the heifers tested were 22.7% (5 of 22) and 8.7% (2 of 23) in winter and spring, respectively. The analysis for presence (PCR) and expression (VTEC-RPEA) of the toxin genes revealed that 37 isolates had and expressed the stx^sub 1^ gene, 14 had and expressed the stx^sub 2^ gene, and five had but did not express the stx^sub 2^ gene (Table 1). The STEC isolates from five : heifers had StX^sub 1^ (Heifers D, F, and G) .J or stx^sub 2^ (Heifers B and C). These isolates were toxic to Vero cells, and the expression of their toxin genes was confirmed by the VTEC-RPEA assay. The four isolates from Heifer E (with the StX^sub 2^ gene) were toxic to Vero cells, but the supernatants of their cultures failed to react with the sensitized latex of the VTEC-RPEA kit (Table 1). The STEC isolate from Heifer A had stx^sub 2^, did not express it (VTEC-RPEA assay), and was not toxic to Vero cells (Table 1). The sequence of the PCR products for StX^sub 1^ matched the published one (Paton et al., 1995), whereas that of the PCR products for StX^sub 2^ did not match (Jackson et al., 1987).

Results of the colony blot (using an anti-O157:H7 antibody) revealed that none of the 56 STEC isolates was O157:H7. This observation is further confirmed by serotyping (Table 1). Serotyping of the STEC isolates revealed that the isolate from Heifer A was E. coli O118:H^sup -^ and the two isolates from Heifer B were E. coli O138:H^sup -^. The STEC isolates from Heifers C, D, E, F, and G, however, were untypable (i.e., unreactive with the O and H monovalent antisera).

Escherichia coli O157:H7 has unique biochemical characteristics [i.e., the lack of sorbitol fermentation and [beta]-glucuronidase activity (tested on the synthetic substrate MUG)]; therefore, it is easier to detect than non-O157:H7 STEG (Willshaw et al, 1997). As a result, testing fecal samples for non-O157:H7 STEC requires the use of other techniques such as cytotoxicity (e.g., verotoxicity) tests and(or) PCR. It is expected that fecal analysis focusing on O157:H7 results in underestimation of STEC occurrence. In the present study, we selected sorbitol-negative isolates that were MUG-negative or MUG-positive to allow for detection of O157:H7 and non-O157:H7 STEQ respectively. Because the antibiotics cefixime and tellurite were not used in the enrichment medium, it is possible that we may have missed some E. coli O157:H7 isolates (Zadik et al., 1993). In our previous studies with cattle (Thran et al., 2001a,b; Hussein et al., 2003a) or sheep (Hussein et al., 2003b), the same enrichment medium was used, and E. coli O157:H7 was detected (Thran et al., ZOOlb; Hussein et al., 2003a). Based on the selection scheme used in this study and in our previous ones (Thran et al, 2001a,b; Hussein et al., 2003a,b), only sorbitol-negative (white colonies on SMAC agar) STEC were identified and characterized. It is worth noting that the 56 STEC isolates were confirmed as sorbitol-negative by using the API 2OE identification system. The STEC isolates that tested positive for [beta]-glucuronidase activity are shown in Table 1.

Examination of the DNA sequence of the PCR products for the Stx2 gene of the 19 STEC isolates from Heifers A, B, C, and E (data not shown) revealed the presence of a mutation in the same location when compared with the published sequence for this gene (Jackson et al., 1987). When compared with stx^sub 2^ sequences in the GenBank, the sequence of our isolates matched a previously submitted one (GenBank Accession: U41241) and was recognized as an E. coll variant shiga-like toxin II gene. It is not known, however, why some of these isolates expressed the stx^sub 2^ gene and some did not (Table 1).

Except for three isolates from Heifers A and B, the remaining STEC were untypeable. Our data (Table 1) revealed the presence of the E. coll serotypes O118:H^sup -^ (one isolate) and O138:H^sup -^ (two isolates). Despite the decreased frequency of human illness outbreaks when compared with E. coll O157:H7, E. coll Ol 18 serotypes should be considered a food safety risk. The Ol 18 serotypes that are known to cause human illnesses worldwide were recently summarized (Bettelheim, 2003) and included O118:H^sup -^ (Table 1), O118:H2, O118:H12, O118:H16, and O118:H30. Of these, O118:H16 and O118:H^sup -^ were isolated from healthy and sick cattle. Escherichia coll O118:H16 was isolated from humans suffering from foodborne illnesses in Spain (Blanco et al., 2003) and Germany (Beutin et al., 2000). Escherichia coli O118:H~ was found to cause HUS in Germany (Beutin et al., 2000), and evidence of its zoonotic transmission from cattle to humans was established (Beutin et al., 2000). Wieler et al. (1998) isolated E. coli O118:H^sup -^ from calves with diarrhea, characterized their virulence factors (i.e., production of verotoxins, ability to cause the attaching and effacing lesion, and production of enterohemorrhagic E. coli hemolysin), and concluded that this serotype is of high virulence potential and can be a major health threat to humans. With regard to E. coli O138:H^sup -^ (Table 1), it does not appear to be a common STEC in cattle, nor has it been associated with human illnesses (Bettelheim, 2003). Members of the Ol38 serogroup, however, have been associated with post-weaning diarrhea and edema in pigs (Parma et al, 2000). Therefore, it is unusual that E. coli O138:H^sup -^ isolates were detected in one of our heifers. The fact that these E. coli O138:H^sup -^ isolates had and expressed one of the virulence factors (e.g., StX^sub 2^ gene) suggests their potential as a food safety risk factor.

In addition to the well-established recognition of E. coli O157:H7 as a foodborne pathogen in the U.S. (USDA-APHIS, 1997), there have been recent concerns with non-O157:H7 STEC as emerging food safety risk factors (CDC, 1995, 2000). These concerns were derived from tracing several outbreaks of human illnesses to non-O157:H7 STEC such as O104:H21 in Montana (CDC, 1995) and members of the serogroup O6 and O26 in Minnesota (Acheson and Keusch, 1996). These outbreaks sug- gest that these pathogens may pose an underestimated threat to public health in the U.S. (Tarr et al., 1996; Acheson et al, 1997). Our previous studies with cattle (Thran et al., 2001a,b; Hussein et al., 2003a) revealed the presence of O157:H^sup -^ and non-O157:H7 (i.e., O6:H49, O6:H^sup -^, O8:H^sup -^, O26:H^sup -^, O39:H^sup -^, O6:H^sup -^, O113:H^sup -^, O116:H^sup -^, and O141:H^sup -^) STEC in Nevada. Of our isolates, E. coli O39:H^sup -^ and O116:H^sup -^ have been isolated from cattle (Montenegro et al., 1990; Blanco et al., 1997; Bettelheim, 2000) but were not associated with outbreaks of human illnesses. However, E. coli O8:H^sup -^ and O141:H^sup -^ have caused major outbreaks of human illnesses (Strockbine et al., 1998; Bettelheim, 2003). A member of the STEC serogroup O105 (i.e., O105:H18) was isolated from healthy cattle and humans with HUS in Spain (Blanco et al., 2003) and Canada (Johnson et al., 1996). Members of the serogroups O6 and Ol 13 also have been associated with human illnesses (Johnson et al., 1996) and were detected in ground beef (Samadpour ct al., 1994). Escherich/a coli O6 was also associated with cases of HUS in France (Bonnet et al., 1998) and of severe diarrhea in Minnesota (Acheson and Keusch, 1996). The E. coli O26 serogroup has been associated with human illnesses in the U.S., and Canada (Acheson and Keusch, 1996), Australia (Desmarchelier, 1997), and Germany (Karch et al., 1997).

In our previous studies, non-O157:H7 serotypes were the majority of STEC detected in Nevada heifers grazing irrigated pastures (Thran et al., 2001a,b) or in cows grazing rangeland forages (Hussein et al., 2003a). In the current study, only non-O157:H7 STEC were detected in beef heifers grazing rangeland forages (Ta- ble 1). Similarly, only non-O157:H7 STEC were detected in sheep grazing rangeland forages (Hussein et al, 2003b). In general, the STEC occurrence rate in the present study was greater for winter (22.7%) than for spring (8.7%). Higher STEC occurrence rates were also detected in winter than in other seasons for beef (15% vs 4%; Thran et al., 2001b) and dairy (9.5% vs 0%; Thran et al, 200Ia) heifers grazing irrigated pastures. Greater occurrence rates were consistently reported for E. coli O157:H7 in the summer than in the winter months (Hancock et al, 1997; Mechie et al., 1997; Van Donkersgoed et al., 1999).

Implications

Based on the health risks associated with the non-O157:H7 STEC detected in this study, it appears that screening cattle for both O157:H7 and non-O157:H7 STEC is critically important. Recent efforts have been devoted to the development of commercial tests that could be used for detecting E. coli O157:H7 at the post-harvest level (e.g., packing plants). These efforts should expand to cover testing for other STEC strains (e.g., E. coli O118:H^sup -^) that are also known to cause human illnesses. At the pre-harvest level, testing cattle for STEC before shipping to slaughter may provide significant benefits to the beef industry. Identification, isolation, and possibly subjecting STEC-positive cattle to pre- and(or) post-harvest control measures would assure safety of their beef and the beef from other animals slaughtered and processed at the same packing facilities.

Acknowledgments

The authors acknowledge the support of the USDA Integrated Research, Education, and Extension Competitive Grants Program (Grant No. 2001-05062). Some of the VTEC-RPEA kits used in this study were kindly provided by Denka Seiken Co., Ltd. (Tokyo, Japan). Appreciation is extended to Ken Conley and John Wilker for the care of the heifers used in this study and for assisting in sample collection at the University of Nevada Gund Research and Demonstration Ranch (Beowawe).

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H. S. HUSSEIN1, B. H. THRAN, and M. R. HALL

Department of Animal Biotechnology, Mail Stop 202, University of Nevada-Reno, Reno 89557

1 To whom correspondence should be addressed: hhussein@agnt1 .ag.unr.edu

Copyright American Registry of Professional Animal Scientists Jun 2004
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