Characterization of Escherichia coli O157:H7 Strains Isolated from Supershedding Cattle

Previous reports have indicated that a small proportion of cattle shedding high levels of Escherichia coli O157:H7 is the main source for transmission of this organism between animals. Cattle achieving a fecal shedding status of 10⁴ CFU of E. coli O157:H7/gram or greater are now referred to as supershedders. The aim of this study was to investigate the contribution of E. coli O157:H7 strain type to supershedding and to determine if supershedding was restricted to a specific set of E. coli O157:H7 strains. Fecal swabs (n = 5,086) were collected from cattle at feedlots or during harvest. Supershedders constituted 2.0% of the bovine population tested. Supershedder isolates were characterized by pulsed-field gel electrophoresis (PFGE), phage typing, lineage-specific polymorphism assay (LSPA), Stx-associated bacteriophage insertion (SBI) site determination, and variant analysis of Shiga toxin, tir, and antiterminator Q genes. Isolates representing 52 unique PFGE patterns, 19 phage types, and 12 SBI clusters were obtained from supershedding cattle, indicating that there is no clustering to E. coli O157:H7 genotypes responsible for supershedding. While being isolated directly from cattle, this strain set tended to have higher frequencies of traits associated with human clinical isolates than previously collected bovine isolates with respect to lineage and tir allele, but not for SBI cluster and Q type. We conclude that no exclusive genotype was identified that was common to all supershedder isolates.     

Escherichia coli O157:H7 in Environments of Culture-Positive Cattle

Outbreaks of Escherichia coli O157:H7 disease associated with animal exhibits have been reported with increasing frequency. Transmission can occur through contact with contaminated haircoats, bedding, farm structures, or water. We investigated the distribution and survival of E. coli O157:H7 in the immediate environments of individually housed, experimentally inoculated cattle by systematically culturing feed, bedding, water, haircoat, and feed bunk walls for E. coli O157:H7 for 3 months. Cedar chip bedding was the most frequently culture-positive environmental sample tested (27/96 or 28.15%). Among these, 12 (44.0%) of positive bedding samples were collected when the penned animal was fecal culture negative. Survival of E. coli O157:H7 in experimentally inoculated cedar chip bedding and in grass hay feed was determined at different temperatures. Survival was longest in feed at room temperature (60 days), but bacterial counts decreased over time. The possibility that urine plays a role in the environmental survival of E. coli O157:H7 was investigated. Cedar chip bedding moistened with sterile water or bovine urine was inoculated with E. coli O157:H7. Bedding moistened with urine supported growth of E. coli O157:H7, whereas inoculated bedding moistened with only water yielded decreasing numbers of bacteria over time. The findings that environmental samples were frequently positive for E. coli O157:H7 at times when animals were culture negative and that urine provided a substrate for E. coli O157:H7 growth have implications for understanding the on-farm ecology of this pathogen and for the safety of ruminant animal exhibits, particularly petting zoos and farms where children may enter animal pens.     

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

Shiga toxin-producing Escherichia coli O157:H7 (O157) are significant foodborne pathogens and pose a serious threat to public health worldwide. The major reservoirs of O157 are asymptomatic cattle which harbor the organism in the terminal recto-anal junction (RAJ). Some colonized animals, referred to as “super-shedders” (SS), are known to shed O157 in exceptionally large numbers (>104 CFU/g of feces). Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. Whole genome sequence analysis of an SS O157 strain (SS17) revealed a genome of 5,523,849 bp chromosome with 5,430 open reading frames and two plasmids, pO157 and pSS17, of 94,645 bp and 37,446 bp, respectively. Comparative analyses showed that SS17 is clustered with spinach-associated O157 outbreak strains, and belongs to the lineage I/II, clade 8, D group, and genotype 1, a subgroup of O157 with predicted hyper-virulence. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci. Phenotypic analyses revealed a distinctive and strongly adherent aggregative phenotype of SS17 on bovine RAJ stratified squamous epithelial (RSE) cells that was conserved amongst other SS isolates. Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism. Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.     

Differences in Virulence among Escherichia coli O157:H7 Strains Isolated from Humans during Disease Outbreaks and from Healthy Cattle

Escherichia coli O157:H7 causes life-threatening outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans and significant economic loss in agriculture and could be a potential agent of bioterrorism. Although the prevalence of E. coli O157:H7 in cattle and other species with which humans have frequent contact is high, human infections are relatively uncommon, despite a low infectious dose. A plausible explanation for the low disease incidence is the possibility that not all strains are virulent in humans. If there are substantial differences in virulence among strains in nature, then human disease may select for high virulence. We used a gnotobiotic piglet model to investigate the virulence of isolates from healthy cattle and from humans in disease outbreaks and to determine the correlation between production of Shiga toxin 1 (Stx1) and Stx2 and virulence. Overall, E. coli O157:H7 strains isolated from healthy cattle were less virulent in gnotobiotic piglets than strains isolated from humans during disease outbreaks. The amount of Stx2 produced by E. coli O157:H7 strains correlated with strain virulence as measured by a reduction in piglet survival and signs of central nervous system disease due to brain infarction. The amount of Stx1 produced in culture was not correlated with the length of time of piglet survival or with signs of central nervous system disease. We suggest that disease outbreaks select for producers of high levels of Stx2 among E. coli O157:H7 strains shed by animals and further suggest that Stx1 expression is unlikely to be significant in human outbreaks.     

Prevalence of Escherichia coli O157:H7 in Gallbladders of Beef Cattle

Gallbladders and rectal contents were collected from cattle (n = 933) at slaughter to determine whether the gallbladder harbors Escherichia coli O157:H7. Both gallbladder mucosal swabs and homogenized mucosal tissues were used for isolation. Only five gallbladders (0.54%) were positive for E. coli O157:H7. Fecal prevalence averaged 7.1%; however, none of the cattle that had E. coli O157:H7 in the gallbladder was positive for E. coli O157:H7 in feces. Therefore, the gallbladder does not appear to be a common site of colonization for E. coli O157:H7 in beef cattle.     

Effect of Cattle Diet on Escherichia coli O157:H7 Acid Resistance

The duration of shedding of Escherichia coli O157 isolates by hay-fed and grain-fed steers experimentally inoculated with E. coli O157:H7 was compared, as well as the acid resistance of the bacteria. The hay-fed animals shed E. coli O157 longer than the grain-fed animals, and irrespective of diet, these bacteria were equally acid resistant. Feeding cattle hay may increase human infections with E. coli O157:H7.     

Genomic Regions Conserved in Lineage II Escherichia coli O157:H7 Strains

Populations of the food- and waterborne pathogen Escherichia coli O157:H7 are comprised of two major lineages. Recent studies have shown that specific genotypes within these lineages differ substantially in the frequencies with which they are associated with human clinical disease. While the nucleotide sequences of the genomes of lineage I strains E. coli O157 Sakai and EDL9333 have been determined, much less is known about the genomes of lineage II strains. In this study, suppression subtractive hybridization (SSH) was used to identify genomic features that define lineage II populations. Three SSH experiments were performed, yielding 1,085 genomic fragments consisting of 811 contigs. Bacteriophage sequences were identified in 11.3% of the contigs, 9% showed insertions and 2.3% deletions with respect to E. coli O157:H7 Sakai, and 23.2% did not have significant identity to annotated sequences in GenBank. In order to test for the presence of these novel loci in lineage I and II strains, 27 PCR primer sets were designed based on sequences from these contigs. All but two of these PCR targets were found in the majority (51.9% to 100%) of 27 lineage II strains but in no more than one (<6%) of the 17 lineage I strains. Several of these linage II-related fragments contain insertions/deletions that may play an important role in virulence. These lineage II-related loci were also shown to be useful markers for genotyping of E. coli O157:H7 strains isolated from human and animal sources.Enterohemorrhagic Escherichia coli is associated with diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans (31). E. coli serotype O157:H7 predominates in epidemics and sporadic cases of enterohemorrhagic E. coli-related infections in the United States, Canada, Japan, and the United Kingdom (12). Cattle are considered the most important reservoir of E. coli O157:H7 (10, 24, 37, 41), and foods contaminated with bovine feces are thought to be the most common source of human infection with this pathogen (27, 33). The two most important virulence factors of the organism are the production of one or more Shiga toxins (Stx) (6, 20, 32) and the ability to attach to and efface microvilli of host intestinal cells (AE). Stx genes are encoded by temperate bacteriophage inserted in the bacterial chromosome, and genes responsible for the AE phenotype are located on the locus of enterocyte effacement (LEE) as well as other pathogenicity islands (4, 17). All E. coli O157:H7 strains also possess a large plasmid which is thought to play a role in virulence (10, 40, 42).Octamer-based genome scanning (OBGS) was first used to show that E. coli O157 strains from the United States and Australia could be subdivided into two genetically distinct lineages (21, 22, 46). While both E. coli O157:H7 lineages are associated with human disease and are isolated from cattle, there is a bias in the host distribution between the two lineages, with a significantly higher proportion of lineage I strains isolated from humans than lineage II strains. Several recent studies have shown that there are inherent differences in gene content and expression between populations of lineage I and lineage II E. coli O157:H7 strains. Lejeune et al. (26) reported that the antiterminator Q gene of the stx₂-converting bacteriophage 933W was found in all nine OBGS lineage I strains examined but in only two of seven lineage II strains, suggesting that there may be lineage-specific differences in toxin production. Dowd and Ishizaki (9) used DNA microarray analysis to examine expression of 610 E. coli O157:H7 genes and showed that lineage I and lineage II E. coli O157:H7 strains have evolved distinct patterns of gene expression which may alter their virulence and their ability to survive in different microenvironments and colonize the intestines of different hosts (9, 28, 38).The observations of lineage host bias have been supported and extended by studies using a six-locus-based multiplex PCR termed the lineage-specific polymorphism assay (LSPA-6) (46). However, Ziebell et al. (48) have recently shown that not all LSPA-6 types within lineage II are host biased; e.g., LSPA-6 type 211111 isolation rates from humans and cattle were significantly different from those of other lineage II LSPA-6 types. Therefore, a clearer definition is required of not only the differences between lineages but also the differences among clonal groups within lineages.The genome sequences of two E. coli O157:H7 strains, Sakai and EDL933 (14, 36), have been determined; however, both of these strains are of lineage I, and there are presently no completed and fully annotated genome sequences available for lineage II strains. In our laboratory, comparative studies utilizing suppression subtractive hybridization (SSH) and comparative genomic hybridization revealed numerous potential virulence factors that are conserved in lineage I strains and that are rare or absent in lineage II strains (42, 47). In this study, we have used SSH to identify genomic regions present in E. coli O157:H7 lineage II strains that are absent from lineage I strains. We wished to examine the distribution of these novel gene segments in E. coli O157:H7 strains and gain insight into their origins and functions. We also attempted to identify molecular markers specific to lineage II strains as well as other markers that would be useful in the genetic subtyping or molecular fingerprinting of E. coli O157:H7 strains in population and epidemiological studies (25). This information may be helpful in the identification of genotypes of the organism associated with specific phenotypes of both lesser and greater virulence (29).     

Derivation of Escherichia coli O157:H7 from Its O55:H7 Precursor

There are 29 E. coli genome sequences available, mostly related to studies of species diversity or mode of pathogenicity, including two genomes of the well-known O157:H7 clone. However, there have been no genome studies of closely related clones aimed at exposing the details of evolutionary change. Here we sequenced the genome of an O55:H7 strain, closely related to the major pathogenic O157:H7 clone, with published genome sequences, and undertook comparative genomic and proteomic analysis. We were able to allocate most differences between the genomes to individual mutations, recombination events, or lateral gene transfer events, in specific lineages. Major differences include a type II secretion system present only in the O55:H7 chromosome, fewer type III secretion system effectors in O55:H7, and 19 phage genomes or phagelike elements in O55:H7 compared to 23 in O157:H7, with only three common to both. Many other changes were found in both O55:H7 and O157:H7 lineages, but in general there has been more change in the O157:H7 lineages. For example, we found 50% more synonymous mutational substitutions in O157:H7 compared to O55:H7. The two strains also diverged at the proteomic level. Mutational synonymous SNPs were used to estimate a divergence time of 400 years using a new clock rate, in contrast to 14,000 to 70,000 years using the traditional clock rates. The same approaches were applied to three closely related extraintestinal pathogenic E. coli genomes, and similar levels of mutation and recombination were found. This study revealed for the first time the full range of events involved in the evolution of the O157:H7 clone from its O55:H7 ancestor, and suggested that O157:H7 arose quite recently. Our findings also suggest that E. coli has a much lower frequency of recombination relative to mutation than was observed in a comparable study of a Vibrio cholerae lineage.     

Association of Escherichia coli O157:H7 with Houseflies on a Cattle Farm

The ecology of Escherichia coli O157:H7 is not well understood. The aims of this study were to determine the prevalence of and characterize E. coli O157:H7 associated with houseflies (HF). Musca domestica L. HF (n = 3,440) were collected from two sites on a cattle farm over a 4-month period and processed individually for E. coli O157:H7 isolation and quantification. The prevalence of E. coli O157:H7 was 2.9 and 1.4% in HF collected from feed bunks and a cattle feed storage shed, respectively. E. coli O157:H7 counts ranged from 3.0 × 10¹ to 1.5 × 10⁵ CFU among the positive HF. PCR analysis of the E. coli O157:H7 isolates revealed that 90.4, 99.2, 99.2, and 100% of them (n = 125) possessed the stx1, stx2, eaeA, and fliC genes, respectively. Large populations of HF on cattle farms may play a role in the dissemination of E. coli O157:H7 among animals and to the surrounding environment.     

Escherichia coli O157:H7 Super-Shedder and Non-Shedder Feedlot Steers Harbour Distinct Fecal Bacterial Communities

Escherichia coli O157:H7 is a major foodborne human pathogen causing disease worldwide. Cattle are a major reservoir for this pathogen and those that shed E. coli O157:H7 at >10⁴ CFU/g feces have been termed “super-shedders”. A rich microbial community inhabits the mammalian intestinal tract, but it is not known if the structure of this community differs between super-shedder cattle and their non-shedding pen mates. We hypothesized that the super-shedder state is a result of an intestinal dysbiosis of the microbial community and that a “normal” microbiota prevents E. coli O157:H7 from reaching super-shedding levels. To address this question, we applied 454 pyrosequencing of bacterial 16S rRNA genes to characterize fecal bacterial communities from 11 super-shedders and 11 contemporary pen mates negative for E. coli O157:H7. The dataset was analyzed by using five independent clustering methods to minimize potential biases and to increase confidence in the results. Our analyses collectively indicated significant variations in microbiome composition between super-shedding and non-shedding cattle. Super-shedders exhibited higher bacterial richness and diversity than non-shedders. Furthermore, seventy-two operational taxonomic units, mostly belonging to Firmicutes and Bacteroidetes phyla, were identified showing differential abundance between these two groups of cattle. The operational taxonomic unit affiliation provides new insight into bacterial populations that are present in feces arising from super-shedders of E. coli O157:H7.     

Genomic typing of Escherichia coli O157:H7 by semi-automated fluorescent AFLP analysis

Escherichia coli serotype O157:H7 isolates were analyzed using a relatively new DNA fingerprinting method, amplified fragment length polymorphism (AFLP). Total genomic DNA was digested with two restriction endonucleases (EcoRI and MseI), and compatible oligonucleotide adapters were ligated to the ends of the resulting DNA fragments. Subsets of fragments from the total pool of cleaved DNA were then amplified by the polymerase chain reaction (PCR) using selective primers that extended beyond the adapter and restriction site sequences. One of the primers from each set was labeled with a fluorescent dye, which enabled amplified fragments to be detected and sized automatically on an automated DNA sequencer. Three AFLP primer sets generated a total of thirty-seven unique genotypes among the 48 E. coli O157:H7 isolates tested. Prior fingerprinting analysis of large restriction fragments from these same isolates by pulsed-field gel electrophoresis (PFGE) resulted in only 21 unique DNA profiles. Also, AFLP fingerprinting was successful for one DNA sample that was not typable by PFGE, presumably because of template degradation. AFLP analysis, therefore, provided greater genetic resolution and was less sensitive to DNA quality than PFGE. Consequently, this DNA typing technology should be very useful for genetic subtyping of bacterial pathogens in epidemiologic studies.     

Prevalence of Escherichia coli O157:H7 in Organically and Naturally Raised Beef Cattle

We determined the prevalence of Escherichia coli O157:H7 in organically and naturally raised beef cattle at slaughter and compared antibiotic susceptibility profiles of the isolates to those of isolates from conventionally raised beef cattle. The prevalences of E. coli O157:H7 were 14.8 and 14.2% for organically and naturally raised cattle, respectively. No major difference in antibiotic susceptibility patterns among the isolates was observed.Many cattle producers have adopted production methods termed niche marketing to meet consumer demand for safe and healthy beef. The two main niches for beef cattle producers are organic and natural production (3). Organic beef cattle production, regulated by the U.S. Department of Agriculture, requires feeding with certified organic feed (16) and raising cattle without the use of antibiotics, hormones, and other veterinary products (3). Guidelines for producers to label the product as “natural” differ among natural beef programs, and such programs are administered and regulated by the company or organization that owns the brand name rather than the U.S. Department of Agriculture (11). Natural production guidelines often include a complete restriction on the use of antibiotics and growth-promoting hormones, but unlike guidelines for organic production, they allow feed from nonorganic sources (11). Escherichia coli O157:H7 is a major food-borne pathogen that causes outbreaks of hemorrhagic enteritis, which often leads to hemolytic uremic syndrome in children and the elderly (10). Cattle are major reservoirs of E. coli O157:H7, which colonizes the hindgut, specifically the rectoanal mucosal region. Cattle feces are the major source of food and water contamination (10). The impact of organic production methods on the prevalence of food-borne pathogens, including E. coli O157:H7 and Campylobacter spp. in dairy cattle (7, 14) and Campylobacter and Salmonella spp. in chickens (6, 19), has been studied previously. However, there is no published study on the prevalence of E. coli O157:H7 in organically and naturally raised beef cattle. Additionally, nothing is known regarding the effects of organic and natural production methods on the antibiotic susceptibilities of E. coli O157:H7 in beef cattle. Our objectives were to determine the prevalence of E. coli O157:H7 in the feces of organically and naturally raised beef cattle at slaughter and compare the antibiotic susceptibilities of isolates from organically, naturally, and conventionally raised beef cattle.Cattle included in this study were from three types of production systems, organic, natural, and conventional. Organically raised beef cattle were from farms that were certified by the National Organic Program (17). The naturally raised beef cattle were from farms that were certified by the All Natural Source Verified Beef Program (17). The collection of samples from these cattle occurred in an abattoir. Samples from conventionally raised cattle from two feedlots were collected in a different abattoir so that the antibiotic susceptibilities of their isolates could be compared with those of isolates from organically and naturally raised cattle. Fecal samples were obtained by cutting open the rectum and spooning out the contents. The mucosa of the rectum was then rinsed with water until free of visible fecal material and swabbed with a sterile foam-tipped applicator (4). The isolation and identification of E. coli O157 and PCR detection of major virulence genes (eae, stx₁, stx₂, hlyA, and fliC) were carried out as described by Reinstein et al. (13). A subset of 60 isolates, 20 (10 from fecal samples and 10 from rectoanal mucosal swabs [RAMS]) from each production system, was randomly chosen to determine the antibiotic susceptibility patterns by the broth microdilution method (9). The antibiotics (all from Sigma-Aldrich) tested were amikacin, amoxicillin (amoxicilline), ampicillin, apramycin, bacitracin, cefoxitin, ceftazidime, ceftriaxone, cephalothin (cefalotin), chloramphenicol, chlortetracycline, ciprofloxacin, enrofloxacin, erythromycin, florfenicol, gentamicin, kanamycin, lincomycin, monensin, nalidixic acid, neomycin, norfloxacin, novobiocin, oxytetracycline, penicillin, rifampin (rifampicin), spectinomycin, streptomycin, tetracycline, tilmicosin, trimethoprim, tylosin, and vancomycin. The MIC was defined as the lowest concentration of an antibiotic that prevented visible growth of the organism. Each concentration of the antibiotic compound was duplicated in the microtiter plate, and the MIC determination was repeated with a different inoculum preparation. Logistic regression was performed using the PROC GENMOD procedure in the SAS system (SAS Institute, Cary, NC) to compare the prevalences of E. coli O157:H7 (with binomial distribution of outcomes) in fecal samples, RAMS samples, and fecal or RAMS samples (overall animal level prevalence). The MICs of antibiotics for E. coli O157:H7 isolates were analyzed using a nonparametric survival test in the PROC LIFETEST program of SAS to determine the effects of the production system (natural, organic, or conventional). Data were right censored when necessary (when the organism was resistant to the highest concentration evaluated). The Wilcoxon test was utilized to determine the effect of the production system on MICs.Samples from a total of 553, 506, and 322 organically, naturally, and conventionally raised cattle, respectively, were collected. In organically raised cattle, the prevalence of E. coli O157:H7 in fecal samples ranged from 0 to 24.4% across sampling days, with an average of 9.3%, and the prevalence in RAMS ranged from 0 to 30.9%, with an average of 8.7% (Fig. ​(Fig.1).1). In naturally raised cattle, the prevalence of E. coli O157:H7 in fecal samples ranged from 0 to 20.3%, with an average of 7.2%, and the prevalence in RAMS ranged from 0 to 23.8%, with an average of 8.9% (Fig. ​(Fig.1).1). In both organically and naturally raised cattle, the prevalence (total) detected by both sampling methods together was greater (P < 0.05) than the prevalence detected by either method alone (Fig. ​(Fig.1).1). Samples (either feces or RAMS) from 36 (11.2%) of 322 conventionally raised feedlot cattle were culture positive for E. coli O157:H7. The fecal prevalence of E. coli O157:H7 was 6.5%, and the prevalence determined by the RAMS sampling method was 7.1%. Most isolates (66.7% from organically raised beef cattle and 77.8% from naturally raised beef cattle) were positive for eae, stx₂, hlyA, and fliC but negative for stx₁. The stx₂ gene was present in 100 and 95% of isolates from organically and naturally raised cattle, respectively. The prevalences of E. coli O157:H7 that we observed in organically and naturally raised beef cattle were similar to the previously reported prevalence in conventionally raised cattle (1). Our study did not include a statistical comparison of the prevalence data because of a number of differences, particularly in diet, among the organic, natural, and conventional production systems. Organically and naturally raised cattle are either required to graze a pasture or fed a forage-based diet. Although conflicting data exist (1), studies have shown that cattle fed a forage diet have both higher levels and longer durations of fecal shedding of E. coli O157:H7 than cattle fed a grain diet (18).Open in a separate windowFIG. 1.Prevalences of E. coli O157:H7 in organically and naturally raised beef cattle at slaughter. For each production system, bars not labeled with the same letter represent significantly different levels at P of <0.05.None of the tested isolates from the three production systems were susceptible to bacitracin, lincomycin, monensin, novobiocin, tilmicosin, tylosin, and vancomycin (MICs > 50 μg/ml). The MICs of 12 antibiotics (amikacin, apramycin, cefoxitin, ceftriaxone, gentamicin, kanamycin, nalidixic acid, neomycin, penicillin, rifampin, streptomycin, and tetracycline) for isolates collected from different production systems were significantly different (P < 0.05). MICs of gentamicin and neomycin for E. coli O157:H7 isolates from conventionally raised cattle were higher (P < 0.05) than those for isolates from naturally and/or organically raised cattle (Table ​(Table1).1). However, MICs of amikacin, apramycin, cefoxitin, ceftriaxone, kanamycin, nalidixic acid, penicillin, rifampin, and tetracycline for isolates from conventionally fed cattle were lower (P < 0.05) than those for isolates from naturally and/or organically raised cattle (Table ​(Table1).1). Among the 60 isolates tested for antibiotic susceptibilities, 6 isolates (10%) were susceptible to all antibiotics included in the study, excluding the seven antibiotics to which all isolates were resistant. Forty-two isolates (70%) were resistant to one antibiotic (MIC, >50 μg or >50 IU/ml), nine isolates (15%) were resistant to two antibiotics, and two isolates (3%) were resistant to five antibiotics. One isolate from the organically raised cattle group was resistant to 10 (amoxicillin, ampicillin, cefoxitin, cephalothin, chloramphenicol, florfenicol, oxytetracycline, penicillin, streptomycin, and tetracycline) of the 26 antibiotics that were inhibitory to other isolates. We have presented the data as the median MICs for each production system. In some instances, the median values were the same but the actual MIC data differed between production systems. This effect occurred because the data were right censored if isolates were not susceptible at 50 μg or 50 IU/ml. If more isolates from a particular production system than from another are censored, it may lead to statistical differences. This pattern justifies the use of survival analysis for this type of data. There were differences between MICs of many antibiotics (cefoxitin, ceftriaxone, gentamicin, nalidixic acid, neomycin, penicillin, rifampin, and tetracycline) for isolates from organically raised cattle and conventionally raised cattle. Similarly, there were differences between MICs of many antibiotics (amikacin, apramycin, ceftriaxone, kanamycin, nalidixic acid, and rifampin) for isolates from naturally raised cattle and conventionally raised cattle. For many of these antibiotics, MICs for isolates from organically or naturally raised cattle were greater than those for isolates from conventionally raised cattle. Resistance genes can be transferred among the enteric pathogen populations in food animals and humans (8), and it is possible that resistance genes from other bacteria in the gastrointestinal system of cattle may be acquired by E. coli O157:H7. For cattle, heavy metals like copper and zinc, which are also antimicrobial, are included in diets at concentrations in excess of the nutritional requirements, often replacing conventional antibiotics, to achieve growth promotion (5). Feeding with metals also results in the emergence of bacterial populations resistant to metals (5), which in some instances may lead to resistance to antibiotics. Mechanisms of resistance to copper at concentrations above those usually tolerated by normal cellular processes have been found on plasmids linked to resistance to antibiotics in some bacteria (5). Therefore, it is possible that isolates from organically or naturally raised cattle that are not exposed to antibiotics still may become resistant to antibiotics.

TABLE 1.

MICs of antimicrobials for E. coli O157:H7 isolates from conventionally, naturally, and organically raised beef cattle

Rapid Determination of Escherichia coli O157:H7 Lineage Types and Molecular Subtypes by Using Comparative Genomic Fingerprinting

In this study, variably absent or present (VAP) regions discovered through comparative genomics experiments were targeted for the development of a rapid, PCR-based method to subtype and fingerprint Escherichia coli O157:H7. Forty-four VAP loci were analyzed for discriminatory power among 79 E. coli O157:H7 strains of 13 phage types (PT). Twenty-three loci were found to maximize resolution among strains, generating 54 separate fingerprints, each of which contained strains of unique PT. Strains from the three previously identified major E. coli O157:H7 lineages, LSPA6-LI, LSPA6-LI/II, and LSPA6-LII, formed distinct branches on a dendrogram obtained by hierarchical clustering of comparative genomic fingerprinting (CGF) data. By contrast, pulsed-field gel electrophoresis (PFGE) typing generated 52 XbaI digestion profiles that were not unique to PT and did not cluster according to O157:H7 lineage. Our analysis identified a subpopulation comprised of 25 strains from a closed herd of cattle, all of which were of PT87 and formed a cluster distinct from all other E. coli O157:H7 strains examined. CGF found five related but unique fingerprints among the highly clonal herd strains, with two dominant subtypes characterized by a shift from the presence of locus fprn33 to its absence. CGF had equal resolution to PFGE typing but with greater specificity, generating fingerprints that were unique among phenotypically related E. coli O157:H7 lineages and PT. As a comparative genomics typing method that is amenable for use in high-throughput platforms, CGF may be a valuable tool in outbreak investigations and strain characterization.     

Escherichia coli O157:H7 Colonization at the Rectoanal Junction of Long-Duration Culture-Positive Cattle

Long-duration consistently Escherichia coli O157:H7 culture-positive cattle were euthanized and necropsied. Tissue and digesta from along the gastrointestinal tract (GIT) were cultured for the bacteria and examined histologically for lymphoid character. E. coli O157:H7 was detected only at the rectoanal junction mucosa and not at any other GIT location.     

Characterization of an Escherichia coli O157:H7 Plasmid O157 Deletion Mutant and Its Survival and Persistence in Cattle

Escherichia coli O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans, and its major reservoir is healthy cattle. An F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other enterohemorrhagic E. coli serotypes. We compared wild-type (WT) E. coli O157:H7 and an isogenic ΔpO157 mutant for (i) growth rates and antibiotic susceptibilities, (ii) survival in environments with various acidity, salt, or heat conditions, (iii) protein expression, and (iv) survival and persistence in cattle following oral challenge. Growth, metabolic reactions, and antibiotic resistance of the ΔpO157 mutant were indistinguishable from those of its complement and the WT. However, in cell competition assays, the WT was more abundant than the ΔpO157 mutant. The ΔpO157 mutant was more resistant to acidic synthetic bovine gastric fluid and bile than the WT. In vivo, the ΔpO157 mutant survived passage through the bovine gastrointestinal tract better than the WT but, interestingly, did not colonize the bovine rectoanal junction mucosa as well as the WT. Many proteins were differentially expressed between the ΔpO157 mutant and the WT. Proteins from whole-cell lysates and membrane fractions of cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Ten differentially expressed ~50-kDa proteins were identified by quadrupole-time of flight mass spectrometry and sequence matching with the peptide fragment database. Most of these proteins, including tryptophanase and glutamate decarboxylase isozymes, were related to survival under salvage conditions, and expression was increased by the deletion of pO157. This suggested that the genes on pO157 regulate some chromosomal genes.     

Dose-Response Envelope for Escherichia coli O157:H7

Escherichia coli O157:H7 is an emerging food and waterborne pathogen in the U.S. and internationally. The objective of this work was to develop a dose-response model for illness by this organism that bounds the uncertainty in the dose-response relationship. No human clinical trial data are available for E. coli O157:H7, but such data are available for two surrogate pathogens: enteropathogenic E. coli (EPEC) and Shigella dysenteriae. E. coli O157:H7 outbreak data provide an initial estimate of the most likely value of the dose-response relationship within the bounds of an envelope defined by beta-Poisson dose-response models fit to the EPEC and S. dysenteriae data. The most likely value of the median effective dose for E. coli O157:H7 is estimated to be approximately 190[emsp4 ]000 colony forming units (cfu). At a dose level of 100[emsp4 ]cfu, the median response predicted by the model is six percent.     

Electrophoretic Mobilities of Escherichia coli O157:H7 and Wild-Type Escherichia coli Strains

The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased.