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).     

Prevalence and Impact of Bacteriophages on the Presence of Escherichia coli O157:H7 in Feedlot Cattle and Their Environment

The relationship between endemic bacteriophages infecting E. coli O157:H7 (referred to as “phage”) and levels of shedding of E. coli O157:H7 by cattle was investigated in two commercial feedlots in southern Alberta, Canada. Between May and November 2007, 10 pens of cattle were monitored by collection of pooled fecal pats, water with sediment from troughs, manure slurry from the pen floor, and rectal fecal samples from individual animals (20 per pen) at two separate times. Bacteriophages infecting E. coli O157:H7 were detected more frequently (P < 0.001) after 18 to 20 h enrichment than by initial screening and were recovered in 239 of 855 samples (26.5% of 411 pooled fecal pats, 23.8% of 320 fecal grab samples, 21.8% of 87 water trough samples, and 94.6% of 37 pen floor slurry samples). Overall, prevalence of phage was highest (P < 0.001) in slurry. Recovery of phage from pooled fecal pats was highest (P < 0.05) in May. Overall recovery did not differ (P > 0.10) between fecal grab samples and pooled fecal pats. A higher prevalence of phage in fecal pats or water trough samples was associated (P < 0.01) with reduced prevalence of E. coli O157:H7 in rectal fecal samples. There was a weak but significant negative correlation between isolation of phage and E. coli O157:H7 in fecal grab samples (r = −0.11; P < 0.05). These data demonstrate that the prevalence of phage fluctuates in a manner similar to that described for E. coli O157:H7. Phage were more prevalent in manure slurry than other environmental sources. The likelihood of fecal shedding of E. coli O157:H7 was reduced if cattle in the pen harbored phage.Bacteriophages are the most abundant biological entities on earth. An estimated 10³⁰ marine bacteriophages are harbored in the ocean, and they significantly influence microbial communities and function (27). As resistance is an increasing challenge in antimicrobial therapy, the antimicrobial nature of bacteriophages is being more intensively studied (13, 15). Bacteriophages naturally inhabit the mammalian gastrointestinal tract (1, 8), and Escherichia coli-infecting bacteriophages are commonly isolated from sewage, hospital wastewater, and fecal samples from humans and animals (3). Ruminants have been shown to shed up to 10⁷ bacteriophage per gram of feces (6), and in humans multiple types of bacteriophage exhibiting activity against E. coli have been isolated from a single fecal sample (7).E. coli O157:H7 is an important zoonotic bacterium carried asymptomatically by cattle and readily isolated from manure, manure slurry, and drinking water in dairies and feedlots (11, 24, 30). Additionally, E. coli O157:H7 shedding by cattle has a seasonal pattern, peaking in the summer months (2, 25). Bacteriophage strains that infect E. coli O157:H7 have also been isolated from animal feces and have shown lytic activity against this bacterium in vivo and in vitro (5, 23, 28, 31). In recent studies, such phages were shown to be widely distributed in cattle and in feces on the pen floor within feedlots (4, 18). However, the relationships between the presence of E. coli O157:H7-infecting bacteriophage in cattle and their environment and the shedding of this bacterium by cattle are largely undefined. Consequently, the aims of the present study were (i) to determine the prevalence of endemic E. coli O157:H7-infecting bacteriophage (referred to as “phage”) in feedlots over a 7-month period and (ii) to compare the presence of phage to the occurrence of E. coli O157:H7 in cattle and their environment.     

rhs Genes Are Potential Markers for Multilocus Sequence Typing of Escherichia coli O157:H7 Strains

DNA sequence-based molecular subtyping methods such as multilocus sequence typing (MLST) are commonly used to generate phylogenetic inferences for monomorphic pathogens. The development of an effective MLST scheme for subtyping Escherichia coli O157:H7 has been hindered in the past due to the lack of sequence variation found within analyzed housekeeping and virulence genes. A recent study suggested that rhs genes are under strong positive selection pressure, and therefore in this study we analyzed these genes within a diverse collection of E. coli O157:H7 strains for sequence variability. Eighteen O157:H7 strains from lineages I and II and 15 O157:H7 strains from eight clades were included. Examination of these rhs genes revealed 44 polymorphic loci (PL) and 10 sequence types (STs) among the 18 lineage strains and 280 PL and 12 STs among the 15 clade strains. Phylogenetic analysis using rhs genes generally grouped strains according to their known lineage and clade classifications. These findings also suggested that O157:H7 strains from clades 6 and 8 fall into lineage I/II and that strains of clades 1, 2, 3, and 4 fall into lineage I. Additionally, unique markers were found in rhsA and rhsJ that might be used to define clade 8 and clade 6. Therefore, rhs genes may be useful markers for phylogenetic analysis of E. coli O157:H7.Escherichia coli O157:H7 was first described in 1983 as the causative agent of a food-borne outbreak attributed to contaminated ground beef patties (35), and it has subsequently emerged as a very important food-borne pathogen. Diseases caused by E. coli O157:H7, such as hemorrhagic colitis and hemolytic uremic syndrome, can be very severe or even life-threatening. Cattle are believed to be the main reservoir for E. coli O157:H7 (5, 15, 41), although other animals may also carry this organism (6, 21). Outbreaks are commonly associated with the consumption of beef and fresh produce that come into contact with bovine feces or feces-contaminated environments, such as food contact surfaces, animal hides, or irrigation water (12, 21, 30, 38).It is well-established that strains of E. coli O157:H7 vary in terms of virulence and transmissibility to humans and that strains differing in these characteristics can be distinguished using DNA-based methods (22, 29, 42). For example, octamer-based genome scanning, which is a PCR approach using 8-bp primers, provided the first evidence that there are at least two lineages of O157:H7, termed lineage I and lineage II (22). Strains classified as lineage I are more frequently isolated from humans than are lineage II strains (42). A later refinement of this classification system was coined the lineage-specific polymorphism assay (LSPA), which classified strains based upon the amplicon size obtained using PCRs targeting six chromosomal regions of E. coli O157:H7 and assigned a six-digit code based upon the pattern obtained (42). Most strains of lineage I grouped into LSPA type 111111, while the majority of lineage II strains fell into LSPA types 211111, 212111, and 222222. More recently, it was suggested that LSPA type 211111 strains comprise a separate group called lineage I/II (45).To gain greater insight into the recent evolution of E. coli O157:H7, a method that is more discriminatory than the LSPA method is desirable. Multilocus sequence typing (MLST) is a method that discriminates between strains of a bacterial species by identifying DNA sequence differences in six to eight targeted genes. Satisfactory MLST schemes exist for other bacterial pathogens (28, 43); however, due to the lack of sequence variations in previously targeted gene markers in E. coli O157:H7 (13, 33), MLST approaches for subtyping this pathogen have been more difficult to develop. More recently, high-throughput microarray and sequencing platforms have been used to identify hundreds of single nucleotide polymorphisms (SNPs) that are useful for discriminating between strains of E. coli O157:H7 during epidemiologic investigations and for drawing phylogenetic inferences (11, 20, 29, 44). Particularly noteworthy, Manning et al. (29) developed a subtyping scheme based upon the interrogation of 32 putative SNP loci. This method separated 528 strains into 39 distinct SNP genotypes, which were grouped into nine statistically supported phylogenetic groups called clade 1 through clade 9. By analyzing the rates of hemolytic uremic syndrome observed in patients infected with strains of clades 2, 7, and 8, it was also concluded that clade 8 strains are more virulent to humans than other strains (29).One drawback of current DNA sequence-based subtyping schemes for E. coli O157:H7 is that they require screening of at least 32 SNP loci. We were interested in asking whether a simpler approach that targets a few informative gene markers could be developed for rapid strain discrimination and phylogenetic determination. A recent analysis of E. coli genomes predicted that rearrangement hot spot (rhs) genes are under the strongest positive selection of all coding sequences analyzed (34). Therefore, we hypothesized that these genes would display significant sequence variations for subtyping O157:H7 strains. The rhs genes were first discovered as elements mediating tandem duplication of the glyS locus in E. coli K-12 (26); however, their function remains unknown. There are nine rhs genes within the genome of the prototypical E. coli O157:H7 strain Sakai, and these genes are designated rhsA, -C, -D, -E, -F, -G, -I, -J, and -K (see Table S1 in the supplemental material) (16). Three of these nine rhs genes, rhsF, -J, and -K, were previously studied by Zhang et al. (44), and a number of SNPs were identified among these genes. However, no studies have been conducted to comprehensively investigate rhs genes as markers in an MLST scheme for subtyping E. coli O157:H7.The primary purpose of the present study was to investigate whether there are sufficient DNA sequence variations among rhs genes to develop an MLST approach for subtyping E. coli O157:H7. In this study, a greater level of DNA sequence variation was observed among rhs genes than in gene markers targeted in previous studies (13, 33). Furthermore, phylogenetic analysis using these rhs genes generally agreed with the established lineage and clade classifications of O157:H7 strains defined previously. We also wanted to determine whether there is a correlation between the lineage classification of O157:H7 strains (42) and the recently proposed clade classification (29). The present study reports evidence that O157:H7 strains from clade 8 are classified as lineage I/II, which is a different lineage from well-studied E. coli O157:H7 outbreak strains, such as EDL933 and Sakai. Therefore, we suggest that outbreaks of O157:H7 are caused by two lineages of this pathogen, lineage I and lineage I/II.     

Transmission and infectious dose of Escherichia coli O157:H7 in swine

Escherichia coli O157:H7 is only occasionally isolated from healthy swine, but some experimentally infected animals will shed the organism in their feces for at least 2 months. Potential explanations for the paucity of naturally occurring infections in swine, as compared to cattle, include a lack of animal-to-animal transmission so that the organism cannot be maintained within a herd, a high infectious dose, or herd management practices that prevent the maintenance of the organism in the gastrointestinal tract. We hypothesized that donor pigs infected with E. coli O157:H7 would transmit the organism to naïve pigs. We also determined the infectious dose and whether housing pigs individually on grated floors would decrease the magnitude or duration of fecal shedding. Infected donor pigs shedding <10⁴ CFU of E. coli O157:H7 per g transmitted the organism to 6 of 12 naïve pigs exposed to them. The infectious dose of E. coli O157:H7 for 3-month-old pigs was approximately 6 × 10³ CFU. There was no difference in the magnitude and duration of fecal shedding by pigs housed individually on grates compared to those housed two per pen on cement floors. These results suggest that swine do not have an innate resistance to colonization by E. coli O157:H7 and that they could serve as a reservoir host under suitable conditions.Escherichia coli O157:H7 and other serotypes of Shiga toxigenic E. coli (STEC) cause an estimated 110,000 cases of human illness yearly in the United States (26). Most cases are thought to occur as a result of the ingestion of contaminated food or water, although direct contacts with animals and person-to-person transmission have also been documented (4). Cattle are considered to be the major reservoir of STEC, and the prevalence of E. coli O157:H7 in the U.S. herd ranges from 2 to 28%, depending on the culture techniques used, the age of the animals, and the season in which samples are collected (10, 12, 15, 17, 29, 33). E. coli O157:H7 has also been recovered from other ruminants such as deer (22, 30) and sheep (24). E. coli O157:H7 has occasionally been isolated from nonruminant animals such as wild birds (32) and raccoons (18), but the bulk of the data suggests that the prevalence of STEC is greater in ruminants than it is in other animals.In the last several years, there have been reports that E. coli O157:H7 has been isolated from healthy swine in Japan, The Netherlands, Sweden, Canada, Norway, and the United States (11, 13, 19, 20, 27; C. L. Gyles, R. Friendship, K. Ziebell, S. Johnson, I. Yong, and R. Amezcua, Proc. 2002 Congr. Int. Pig Vet. Soc., abstr. 191). The prevalence of the organism in these studies is generally low (0.1 to 6%), and no human outbreaks have been specifically traced back to pork, although sausage containing both beef and pork was implicated as the source of human infection in at least one outbreak (28). In Chile, the prevalence of E. coli O157:H7 reported from pigs (10.8%) was greater than that reported from cattle (2.9%), suggesting that swine may be an important source of this organism in some countries (3). Previously, we have shown that some market-weight pigs experimentally infected with E. coli O157:H7 will shed the organism for at least 2 months (2). These animals do not become clinically ill, and the magnitude and duration of fecal shedding of E. coli O157:H7 are reminiscent of those seen in experimentally infected ruminants (6, 7). This suggests that swine have the biological potential to emerge as a reservoir for E. coli O157:H7 and other STEC strains pathogenic for humans. In order for swine to serve as a reservoir host, not only must the organism colonize the gastrointestinal tract of individual animals, it must also be transmitted from colonized animals to naïve animals to be maintained within the herd. In addition, the infectious dose must be of such a magnitude that a natural infection could be perpetuated within the herd. We hypothesized that E. coli O157:H7 would be transmitted from infected donor pigs to naïve pigs at levels that could be sustained in a natural infection. In addition, we determined the infectious dose of in vitro-grown E. coli O157:H7 for 3-month-old pigs and determined whether housing pigs individually on raised decks or in groups on cement floors affected the magnitude and duration of fecal shedding in infected animals.(A preliminary report of this work was presented at the International Symposium on Shiga Toxin-Producing E. coli, Kyoto, Japan, 2000, and Edinburgh, Scotland, 2003.)     

In Vivo and Ex Vivo Evaluations of Bacteriophages e11/2 and e4/1c for Use in the Control of Escherichia coli O157:H7

This study investigated the effect of bacteriophages (phages) e11/2 and e4/1c against Escherichia coli O157:H7 in an ex vivo rumen model and in cattle in vivo. In the ex vivo rumen model, samples were inoculated with either 10³ or 10⁶ CFU/ml inoculum of E. coli O157:H7 and challenged separately with each bacteriophage. In the presence of phage e11/2, the numbers of E. coli O157:H7 bacteria were significantly (P < 0.05) reduced to below the limit of detection within 1 h. Phage e4/1c significantly (P < 0.05) reduced E. coli O157:H7 numbers within 2 h of incubation, but the number of surviving E. coli O157:H7 bacteria then remained unchanged over a further 22-h incubation period. The ability of a phage cocktail of e11/2 and e4/1c to reduce the fecal shedding of E. coli O157:H7 in experimentally inoculated cattle was then investigated in two cattle trials. Cattle (yearlings, n = 20 for trial one; adult fistulated cattle, n = 2 for trial two) were orally inoculated with 10¹⁰ CFU of E. coli O157:H7. Animals (n = 10 for trial one; n = 1 for trial two) were dosed daily with a bacteriophage cocktail of 10¹¹ PFU for 3 days postinoculation. E. coli O157:H7 and phage numbers in fecal and/or rumen samples were determined over 7 days postinoculation. E. coli O157:H7 numbers rapidly declined in all animals within 24 to 48 h; however, there was no significant difference (P > 0.05) between the numbers of E. coli O157:H7 bacteria shed by the phage-treated or control animals. Phages were recovered from the rumen but not from the feces of the adult fistulated animal in trial two but were recovered from the feces of the yearling animals in trial one. While the results from the rumen model suggest that phages are effective in the rumen, further research is required to improve the antimicrobial effectiveness of phages for the elimination of E. coli O157:H7 in vivo.Escherichia coli O157:H7 has become a worldwide public health concern since it was first identified as a human pathogen in 1982 (31). This pathogen has a very low infectious dose (approximately 10 cells) in humans, and symptoms of infection range from watery diarrhea to hemorrhagic colitis and hemolytic uremic syndrome, and in some cases, death (22, 39). Ruminants are recognized as reservoirs for this pathogen and are the most common sources for food-borne outbreaks (8, 13, 25). It has been reported that the occurrence of E. coli O157:H7 in the feces and, in particular, the hide of cattle is a significant source of the pathogen on the carcass and in derived meat products (11, 12, 25). The control of this pathogen within the animal is difficult, because carriage in ruminants is asymptomatic and shedding can be intermittent and seasonal (12, 19).Research has highlighted the necessity for preharvest intervention strategies to control or reduce E. coli O157:H7 in the food chain (17, 18). Successful strategies to reduce the carriage of E. coli O157:H7 in ruminant animals could potentially reduce the risk of human exposure to this pathogen. There are currently no effective and reliable commercially available intervention strategies to control the carriage of E. coli O157:H7 in ruminants. However, research in this area is increasing, and numerous agents, such as vaccines, probiotics, and bacteriophages (phages), are being evaluated (15, 17, 18). The use of phages for the control of food-borne pathogens in the food chain is desirable, as they are natural, nontoxic viruses that target only specific bacteria (2) and are already being used in human and veterinary medicine, particularly prior to antibiotics (6, 14, 15, 30, 37). Many studies have investigated the use of different phages for the control of E. coli O157:H7 in various animals, including mice, calves, and sheep (4, 5, 35, 37, 41). Although the results between studies vary, some have reported the successful reduction of E. coli O157:H7 levels in animals (4), and one study has resulted in a U.S. patent (41). There are very few commercially available phage products to date, but research indicates promising outcomes for the use of phages for the control of E. coli O157:H7 within the food chain.The E. coli O157:H7-specific phages e11/2 and e4/1c were isolated from bovine slurry in a previous study (26) and have the potential to be used as biocontrol agents for E. coli O157:H7. Both phages have been found to be active against E. coli O157:H7 in a number of relevant test conditions involving different pHs, water activity, and temperatures (B. Coffey, L. Rivas, G. Duffy, A. Coffey, R. P. Ross, and O. McAuliffe, unpublished data). In addition, whole-genome sequencing revealed that neither phage encodes undesirable properties, such as virulence factors, that would hinder its use as a biocontrol agent for E. coli O157:H7 (B. Coffey, G. O''Flynn, A. Coffey, O. O''Sullivan, O. McAuliffe, and R. P. Ross, unpublished data). The objective of the present study was, first, to evaluate the effect of phages e4/1c and e11/2 against inoculated E. coli O157:H7 in an ex vivo model rumen system, and second, to assess the ability of a phage cocktail (e11/2 and e4/1c) to reduce the shedding of E. coli O157:H7 in experimentally inoculated cattle. Findings from ex vivo studies determined our phages to be effective against E. coli O157:H7 in a model rumen system; however, complete eradication of E. coli O157:H7 from cattle was not achieved.     

Indirect transmission of Escherichia coli O157:H7 occurs readily among swine but not among sheep

Transmission of Escherichia coli O157:H7 among reservoir animals is generally thought to occur either by direct contact between a naïve animal and an infected animal or by consumption of food or water containing the organism. Although ruminants are considered the major reservoir, there are two reports of human infections caused by E. coli O157:H7 linked to the consumption of pork products or to the contamination of fresh produce by swine manure. The objective of this study was to determine whether E. coli O157:H7 could be transmitted to naïve animals, both sheep and swine, that did not have any direct contact with an infected donor animal. We recovered E. coli O157:H7 from 10/10 pigs with nose-to-nose contact with the infected donor or animals adjacent to the donor and from 5/6 naïve pigs that were penned in the same room as the donor pig but 10 to 20 ft away. In contrast, when the experiment was repeated with sheep, E. coli O157:H7 was recovered from 4/6 animals that had nose-to-nose contact with the infected donor or adjacent animals and from 0/6 naïve animals penned 10 to 20 ft away from the donor. These results suggest that E. coli O157:H7 is readily transmitted among swine and that transmission can occur by the creation of contaminated aerosols.Escherichia coli O157:H7 infections are an important cause of food-borne illness in much of the world. Human disease usually results from the contamination of food or water by ruminant manure, and cattle are considered to be the primary reservoir of Shiga toxin-producing E. coli, including serotype O157:H7. Over the last several years E. coli O157:H7 has been recovered from small numbers of healthy pigs in Japan (17), Canada (11), Sweden (9), and the United States (10, 14). Recently, a small cluster of human infections caused by E. coli O157:H7 were traced back to dry fermented pork salami as the source (6). In addition, a large outbreak of human cases in the United States was linked to spinach potentially contaminated by both feral swine and cattle manure (12). E. coli O157:H7 can be carried by experimentally infected swine for at least 2 months (3, 4), and we have shown that transmission between naïve animals penned with an infected donor occurs freely (3, 4, 8). The objective of the current study was to determine whether or not E. coli O157:H7 could be transmitted to naïve animals that did not have any direct contact with an infected donor animal.(A preliminary report of this work was presented at the Annual Meeting of the Food Safety Consortium, Fayetteville, AR, and at the Annual Meeting of the American Society for Microbiology, Toronto, Canada, 2007.)     

Escherichia coli O157:H7 Strain Origin,Lineage, and Shiga Toxin 2 Expression Affect Colonization of Cattle

Enterohemorrhagic Escherichia coli O157:H7 has evolved into an important human pathogen with cattle as the main reservoir. The recent discovery of E. coli O157:H7-induced pathologies in challenged cattle has suggested that previously discounted bacterial virulence factors may contribute to the colonization of cattle. The objective of the present study was to examine the impact of lineage type, cytotoxin activity, and cytotoxin expression on the amount of E. coli O157:H7 colonization of cattle tissue and cells in vitro. Using selected bovine- and human-origin strains, we determined that lineage type predicted the amount of E. coli O157:H7 strain colonization: lineage I > intermediate lineages > lineage II. All E. coli O157:H7 strain colonization was dose dependent, with threshold colonization at 10³ to 10⁵ CFU and maximum colonization at 10⁷ CFU. We also determined that an as-yet-unknown factor of strain origin was the most dominant predictor of the amount of strain colonization in vitro. The amount of E. coli O157:H7 colonization was also influenced by strain cytotoxin activity and the inclusion of cytotoxins from lineage I or intermediate lineage strains increased colonization of a lineage II strain. There was a higher level of expression of the Shiga toxin 1 gene (stx₁) in human-origin strains than in bovine-origin strains. In addition, lineage I strains expressed higher levels of the Shiga toxin 2 gene (stx₂). The present study supports a role for strain origin, lineage type, cytotoxin activity, and stx₂ expression in modulating the amount of E. coli O157:H7 colonization of cattle.Enterohemorrhagic Escherichia coli O157:H7 is a bacterium that causes serious human disease outbreaks through the consumption of contaminated food or water (39). Mature cattle are considered the primary reservoir for E. coli O157:H7 and historically were reported to have no symptoms or pathologies (17, 23, 38); this was attributed both to a lack of receptors for a critical E. coli O157:H7 virulence factor, Shiga toxin 1 (Stx1 [29]), and to a differential expression of type III protein secretion system effector molecules such as EspA, EspD, and Iha (25, 30) in cattle compared to humans. In 2008, it was established for the first time that E. coli O157:H7 causes mild to severe intestinal pathology in persistent shedding cattle (5, 26) and that the secreted cytotoxins enhanced E. coli O157:H7 colonization of intestinal tissues of cattle (6). This suggested that cattle were susceptible to E. coli O157:H7 infection and that previously discounted virulence factors could influence the amount of colonization in cattle.Three distinct E. coli O157:H7 lineages have been identified based on the lineage specific polymorphism assay (LSPA-6) that suggests both the evolutionary history of the strain and their propensity to be present among animals, the environment, and clinical human isolates (21, 22, 24, 33, 40, 42). Typically, two predominant lineages have been described, lineages I and II (22, 40) and, more recently, intermediate lineages that have characteristics of lineage I and/or II have been reported at higher frequency among cattle (34). Although all E. coli O157:H7 lineages have been isolated from feedlot cattle, the predominant recovery of lineage I from clinical human illnesses suggests that this particular lineage type has unique expression patterns that may contribute to its preferential colonization of humans. There is some evidence to suggest that lineage I strains do not express certain virulence factors in bovine hosts, whereas other factors such as cytotoxins are expressed equally irrespective of host (30). One virulence factor associated with all lineages is the bacterium''s ability to form intimate attaching-and-effacing lesions or colonization sites in the ilea of susceptible animals (28). The amount of colonization is enhanced by the expression of Shiga toxin 2 (Stx2) through both an increase in the expression of alternative non-TIR (translocated intimin receptor) colonization sites (31) and toxicity to the absorptive epithelial cells (32). In cattle, attaching-and-effacing lesions are also formed (5), and Stx2 increases colonization but is not cytotoxic to epithelial cells from the jejuna and descending colons of cattle (4). Differential expression of stx₂ among E. coli O157:H7 lineages is also linked to the increased pathogenicity of lineage I strains in humans (25), and this may affect cattle similarly. Together, this information suggests that at least some similar virulence factors affecting E. coli O157:H7 colonization in humans also function in cattle.In order to gain a better understanding of the factors modulating E. coli O157:H7 colonization in cattle, we compared the ability of lineage I, lineage II, and intermediate lineages isolated from human sources to colonize the jejunum tissue and a colonic cell line from cattle. We hypothesized that the bovine colonic cell line could be used as a model system to reflect E. coli O157:H7 colonization of tissue. To confirm the value of this model, the role of strain origin in colonization of cattle was examined. In order to understand the differences in colonization associated with lineage and strain origins, we assessed cytotoxin expression, secreted cytotoxin activity, and cytotoxin-induced changes in E. coli O157:H7 colonization. Given the known lack of Stx1 activity in cattle, we examined the effects of LSPA-6 genotype, strain origin (human versus bovine), and cytotoxin activity on E. coli O157:H7 colonization of cattle.     

Rapid Microarray-Based Genotyping of Enterohemorrhagic Escherichia coli Serotype O156:H25/H?/Hnt Isolates from Cattle and Clonal Relationship Analysis

Since enterohemorrhagic Escherichia coli (EHEC) isolates of serogroup O156 have been obtained from human diarrhea patients and asymptomatic carriers, we studied cattle as a potential reservoir for these bacteria. E. coli isolates serotyped by agglutination as O156:H25/H−/Hnt strains (n = 32) were isolated from three cattle farms during a period of 21 months and characterized by rapid microarray-based genotyping. The serotyping by agglutination of the O156 isolates was not confirmed in some cases by the results of DNA-based serotyping as only 25 of the 32 isolates were conclusively identified as O156:H25. In the multilocus sequence typing (MLST) analysis, all EHEC O156:H25 isolates were characterized as sequence type 300 (ST300) and ST688, which differ by a single-nucleotide exchange in the purA gene. Oligonucleotide microarrays allow simultaneous detection of a wider range of EHEC-associated and other E. coli virulence markers than other methods. All O156:H25 isolates showed a wide spectrum of virulence factors typical for EHEC. The stx₁ genes combined with the EHEC hlyA (hlyA_EHEC) gene, the eae gene of the ζ subtype, as well as numerous other virulence markers were present in all EHEC O156:H25 strains. The behavior of eight different cluster groups, including four that were EHEC O156:H25, was monitored in space and time. Variations in the O156 cluster groups were detected. The results of the cluster analysis suggest that some O156:H25 strains had the genetic potential for a long persistence in the host and on the farm, while other strains did not. As judged by their pattern of virulence markers, E. coli O156:H25 isolates of bovine origin may represent a considerable risk for human infection. Our results showed that the miniaturized E. coli oligonucleotide arrays are an excellent tool for the rapid detection of a large number of virulence markers.Shiga toxin-producing Escherichia coli (STEC) strains comprise a group of zoonotic enteric pathogens (45). In humans, infections with some STEC serotypes may result in hemorrhagic or nonhemorrhagic diarrhea, which can be complicated by the hemolytic uremic syndrome (HUS) (32). These STEC strains are also designated enterohemorrhagic Escherichia coli (EHEC). Consequently, EHEC strains represent a subgroup of STEC with high pathogenic potential for humans. Although E. coli O157:H7 is the most frequent EHEC serotype implicated in HUS, other serotypes can also cause this complication. Non-O157:H7 EHEC strains including serotypes O26:H11/H−, O103:H2/H−, O111:H8/H10/H−, and O145:H28/H25/H− and sorbitol-fermenting E. coli O157:H− isolates are present in about 50% of stool cultures from German HUS patients (10, 42). However, STEC strains that cause human infection belong to a large number of E. coli serotypes, although a small number of STEC isolates of serogroup O156 were associated with human disease (7). Strains of the serotypes O156:H1/H8/H21/H25 were found in human cases of diarrhea or asymptomatic infections (9, 22, 25, 26). The detection of STEC of serogroup O156 from healthy and diseased ruminants such as cattle, sheep, and goats was reported by several authors (1, 11-13, 21, 39, 46, 50, 52). Additional EHEC-associated virulence genes such as stx, eae, hlyA_EHEC, or nlaA were found preferentially in the serotypes O156:H25 and O156:H− (11-13, 21, 22, 50, 52).Numerous methods exist for the detection of pathogenic E. coli, including genotypic and phenotypic marker assays for the detection of virulence genes and their products (19, 47, 55, 57). All of these methods have the common drawback of screening a relatively small number of determinants simultaneously. A diagnostic DNA microarray based on the ArrayTube format of CLONDIAG GmbH was developed as a viable alternative due to its ability to screen multiple virulence markers simultaneously (2). Further microarray layouts working with the same principle but different gene targets were developed for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria (5) and for the rapid DNA-based serotyping of E. coli (4). In addition, a protein microarray for E. coli O serotyping based on the ArrayTube format was described by Anjum et al. (3).The aim of our study was the molecular genotyping of bovine E. coli field isolates of serogroup O156 based on miniaturized E. coli oligonucleotide arrays in the ArrayStrip format and to combine the screening of E. coli virulence markers, antimicrobial resistance genes, and DNA serotyping targets, some of which were partially described previously for separate arrays (2, 4, 5). The epidemiological situation in the beef herds from which the isolates were obtained and the spatial and temporal behavior of the clonal distribution of E. coli serogroup O156 were analyzed during the observation period. The potential risk of the isolates inducing disease in humans was assessed.     

Adherence of Escherichia coli O157:H7 Mutants In Vitro and in Ligated Pig Intestines

There are contradictory literature reports on the role of verotoxin (VT) in adherence of enterohemorrhagic Escherichia coli O157:H7 (O157 EHEC) to intestinal epithelium. There are reports that putative virulence genes of O island 7 (OI-7), OI-15, and OI-48 of this pathogen may also affect adherence in vitro. Therefore, mutants of vt2 and segments of OI-7 and genes aidA₁₅ (gene from OI-15) and aidA₄₈ (gene from OI-48) were generated and evaluated for adherence in vitro to cultured human HEp-2 and porcine jejunal epithelial (IPEC-J2) cells and in vivo to enterocytes in pig ileal loops. VT2-negative mutants showed significant decreases in adherence to both HEp-2 and IPEC-J2 cells and to enterocytes in pig ileal loops; complementation only partially restored VT2 production but fully restored the adherence to the wild-type level on cultured cells. Deletion of OI-7 and aidA₄₈ had no effect on adherence, whereas deletion of aidA₁₅ resulted in a significant decrease in adherence in pig ileal loops but not to the cultured cells. This investigation supports the findings that VT2 plays a role in adherence, shows that results obtained in adherence of E. coli O157:H7 in vivo may differ from those obtained in vitro, and identified AIDA-15 as having a role in adherence of E. coli O157:H7.Escherichia coli O157:H7 is the prototypical enterohemorrhagic E. coli (EHEC) strain and is the most common serotype associated with large outbreaks and sporadic cases of hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS) (25). It is well established that EHEC O157:H7 can colonize the intestine of humans and animals and that adherence to intestinal epithelial cells occurs through the formation of attaching-and-effacing (AE) lesions, which is a critical early step in infection. Some researchers have suggested that EHEC uses fimbriae to make the initial contact with epithelial cells, prior to intimate attachment mediated by locus of enterocyte effacemen-encoded proteins (15). Several potential adherence factors of EHEC O157:H7 have been described, but only the outer membrane protein intimin has been demonstrated to play a role in intestinal colonization in animal models (16). Intimin mediates the intimate adherence component of the AE lesion by binding to the translocated intimin receptor Tir, resulting in close attachment of the bacteria to the host cell membrane (17). Intimin can also bind to β1 integrins and nucleolin on host cells (9, 36). Severe damage due to infection with EHEC is attributable to the cytotoxic verotoxin (VT), which damages epithelial and endothelial cells, leading to bloody diarrhea and HUS (16). Several investigators have reported that VT does not play a role in colonization of the intestine (2, 4, 34). However, Robinson et al. (30) reported recently that VT enhances adherence to epithelial cells and colonization of the mouse intestine by E. coli O157:H7. Therefore, the present study examined the involvement of VT in adherence in vitro and in vivo.Several putative virulence genes have been identified in O islands (OIs) in EHEC O157:H7 strain EDL 933 (26), including those encoding Iha and AIDA-I in OI-43/48, AIDA-I in OI-15, and a ClpB chaperone protein and a putative macrophage toxin in OI-7 (26, 27). OI-7 also contains many unknown open reading frames (ORFs) whose function in the pathogenesis of EHEC O157:H7 has not been investigated. Iha, an adherence-conferring outer membrane protein similar to IrgA (the product of iron-regulated gene A) (38), is a virulence factor in uropathogenic E. coli strain CFT073 (14). AIDA-I, encoded by aidA, was first identified in EPEC and confers the capacity for diffuse adhesion of the bacteria to epithelial cells (1). AIDA-I-like adhesins from OI-15 and OI-43/48 show 55% and 68% homology, respectively, to the AIDA-I of EPEC (26, 27). All three AIDA proteins show characteristics of an autotransporter membrane protein with a β-barrel structure (20), which is exposed at the surface of the bacteria (13). These observations suggest that the two homologs of AIDA-I may also function as adhesins in EHEC O157:H7; however, the roles of the AIDA-I-like adhesins in EHEC have yet to be determined.EHEC O157:H7 has been isolated from pigs, and conventional pigs are a permissive host and therefore a potential reservoir for human infection with EHEC O157:H7 (8). One recent family outbreak was associated with pork salami (3). Pigs are highly relevant models for the study of virulence of EHEC O157:H7 in humans and have been extensively used to characterize putative virulence factors and to investigate the pathogenesis of EHEC O157:H7 and other verotoxigenic E. coli strains (6, 11, 21). The present study was designed to examine VT2-negative mutants, OI-7 deletions, and aidA knockouts from OI-15 and OI-48 of EHEC O157:H7 in vitro and in the pig intestines for their roles in adherence.     

Inclusion of Dried or Wet Distillers' Grains at Different Levels in Diets of Feedlot Cattle Affects Fecal Shedding of Escherichia coli O157:H7

Our objectives were to evaluate the prevalence of Escherichia coli O157:H7 in cattle fed diets supplemented with 20 or 40% dried distillers'' grains (DG) (DDG) or wet DG (WDG) and assess whether removing DG from diets before slaughter affected fecal shedding of E. coli O157:H7. Eight hundred forty steers were allocated to 70 pens (12 steers/pen). Treatments were no DG (control), 20% DDG or WDG, and 40% DDG or WDG, and each was replicated in 14 pens. In phase 1, eight floor fecal samples were collected from each pen every 2 weeks for 12 weeks for isolation of E. coli O157:H7 and detection of high shedders. In phase 2, half of the pens with DG were transitioned to the no-DG control diet, and pen floor fecal samples were collected weekly from all pens for 4 weeks. During phase 1, prevalence of E. coli O157:H7 was 20.8% and 3.2% for high shedders. The form of DG had no significant effect on fecal E. coli O157:H7 shedding. The prevalence levels of E. coli O157:H7 and the numbers of high shedders were not different between diets with 0 or 20% DG; however, cattle fed 40% DG had a higher prevalence and more high shedders than cattle fed 0 or 20% DG (P ≤ 0.05). During phase 2, overall and high-shedder prevalence estimates were 3.3% and <0.1%, respectively, and there were no differences between those for different DG forms and inclusion levels or when DG was removed from diets. The form of DG had no impact on E. coli O157:H7; however, fecal shedding was associated with the DG inclusion level.Cattle are asymptomatic reservoirs for Escherichia coli O157:H7, a food-borne pathogen associated with gastrointestinal disease in thousands of Americans each year. The organism colonizes the hindgut of cattle (18, 27) and is shed in cattle feces. Once shed, E. coli O157:H7 can contaminate food and water, creating a food safety risk (20). Contamination of beef products occurs during slaughter and is associated with the prevalence of E. coli O157:H7 in feces and on the hides of cattle at harvest (5, 8, 12).The prevalence of E. coli O157:H7 in cattle is associated with many factors, including season, geographic location, and diet. Previous work has shown that cattle fed diets containing distillers'' grains (DG), an ethanol fermentation coproduct, have a higher prevalence of E. coli O157:H7 than cattle fed diets without DG (10, 28). Distillers'' grains are a valuable feed commodity for cattle producers, and use of these coproducts has increased with the expansion of the ethanol industry (14, 17). Distillers'' grains for use in cattle diets are available in wet (WDG) or dry (DDG) form. The association between feeding DG and E. coli O157:H7 prevalence has been shown with both forms (10, 28), but no study has directly compared the two forms. The levels of DG supplementation in cattle diets generally range from 10 to 50% (dry matter basis) depending on whether the coproduct is used as a protein or energy source. As a protein supplement, DG is included at 10 to 15%; as an energy source, the DG level is generally dictated by coproduct availability and grain price (14). There is some indication that E. coli O157:H7 prevalence is different for cattle fed different levels of DG (19). However, no study has specifically evaluated the relationship between E. coli O157:H7 prevalence and DG inclusion level. Evaluation of these two factors (form and inclusion level) is important for furthering our understanding of the association between DG and E. coli O157:H7 in cattle.We also were interested in determining whether removing the DG component of the diet would lower fecal prevalence of E. coli O157:H7. Such a strategy may lead to potential mitigation options and would provide further evidence of a positive association between feeding DG and E. coli O157:H7 prevalence in cattle. In this two-phase study, our objectives were to (i) concurrently evaluate the effect of DG inclusion level and form on E. coli O157:H7 prevalence in feedlot cattle and (ii) determine if removing DG from cattle diets subsequently reduces the fecal prevalence of E. coli O157:H7.     

Recovery and Detection of Escherichia coli O157:H7 in Surface Water,Using Ultrafiltration and Real-Time PCR

Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) outbreaks have revealed the need for improved analytical techniques for environmental samples. Ultrafiltration (UF) is increasingly recognized as an effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. This study describes the application of hollow-fiber UF as the primary step for concentrating EHEC O157:H7 seeded into 40-liter samples of surface water, followed by an established culture/immunomagnetic-separation (IMS) method and a suite of real-time PCR assays. Three TaqMan assays were used to detect the stx1, stx2, and rfbE gene targets. The results from this study indicate that approximately 50 EHEC O157:H7 cells can be consistently recovered from a 40-liter surface water sample and detected by culture and real-time PCR. Centrifugation was investigated and shown to be a viable alternative to membrane filtration in the secondary culture/IMS step when water quality limits the volume of water that can be processed by a filter. Using multiple PCR assay sets to detect rfbE, stx1, and stx2 genes allowed for specific detection of EHEC O157:H7 from strains that do not possess all three genes. The reported sample collection and analysis procedure should be a sensitive and effective tool for detecting EHEC O157:H7 in response to outbreaks of disease associated with contaminated water.Several highly publicized outbreaks of gastrointestinal diseases caused by enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) have highlighted the threat this pathogen poses to public health (1, 2, 3, 14). Although the predominant mode of transmission to humans appears to be contaminated meat or meat products, there have been a number of outbreaks associated with contaminated water (18). Microbiological, epidemiological, and environmental studies have found an association between EHEC O157:H7 outbreaks and recreational water, drinking water, crop irrigation, and wastewater (1, 2, 14). These investigations have also revealed that enhanced rapid analytical techniques are needed to improve the speed and effectiveness of these types of investigations.Hollow-fiber ultrafiltration (UF) is a sampling technique that is emerging as an option for recovering diverse microbes from large-volume water samples (8, 9, 12, 13, 15). There have been reports of the successful application of UF for surface water as well as for other E. coli strains (8, 13), but additional data are needed to evaluate the robustness of UF for surface water and its ability to effectively concentrate EHEC O157:H7 in the presence of background microbes. The presence of competitive microbes has been shown to significantly alter the growth rate and maximal density of EHEC O157:H7 in broth culture (5).EHEC O157:H7 is generally detected in water samples by using membrane filtration, selective broth enrichment, immunomagnetic-separation (IMS), and isolation on selective agar culture plates, followed by confirmatory tests such as PCR or serological tests (6, 7). However, sensitive detection of EHEC O157:H7 in surface waters can be difficult due to high levels of competing background microorganisms (7). Membrane filtration can also limit the volume processed for turbid surface waters due to filter clogging. Centrifugation is an alternative to membrane filtration and has an advantage of not being subject to potential sample volume processing constraints for turbid water samples, so the technique could potentially increase the sensitivity of detection. A number of PCR assays have been developed for detection of EHEC O157:H7 that target a variety of virulence genes (17). Testing multiple gene targets is necessary for accurate detection because certain non-EHEC O157:H7 serotypes and other bacterial species are known to possess the target genes; therefore, the isolate cannot be determined to be EHEC O157:H7 unless multiple assays show a positive signal (19).The goals of this study were to evaluate (i) the effectiveness of a previously reported UF method (8) for application to recovering EHEC O157:H7, (ii) the effectiveness of the culture/IMS technique performed in conjunction with primary UF concentration, (iii) the effectiveness of centrifugation as an alternative for membrane filtration in the culture/IMS method, and (iv) the ability of three previously reported real-time PCR assays to accurately detect EHEC O157:H7 in surface waters (16, 17).     

Rapid Affinity Immunochromatography Column-Based Tests for Sensitive Detection of Clostridium botulinum Neurotoxins and Escherichia coli O157

Existing methods for detection of food-borne pathogens and their toxins are frequently time-consuming, require specialized equipment, and involve lengthy culture procedures and/or animal testing and are thus unsuitable for a rapid response to an emergency public health situation. A series of simple and rapid affinity immunochromatography column (AICC) assays were developed to detect Clostridium botulinum neurotoxin types A, B, E, and F and Escherichia coli O157 in food matrices. Specifically, for milk, grape juice with peach juice, and bottled water, the detection limit for the botulinum neurotoxin type A complex was 0.5 ng. Use of this method with a 10-ml sample would therefore result in a detection limit of 50 pg ml^−l. Thus, this assay is approximately 2 orders of magnitude more sensitive than a comparable lateral-flow assay. For botulinum neurotoxin complex types B, E, and F, the minimum detection limit was 5 ng to 50 ng. Sensitive detection of E. coli O157 was achieved, and the detection limit was 500 cells. The AICC test was also shown to be specific, rapid, and user friendly. This test takes only 15 to 30 min to complete without any specialized equipment and thus is suitable for use in the field. It has the potential to replace existing methods for presumptive detection of botulinum neurotoxin types A, B, E, and F and E. coli O157 in contaminated matrices without a requirement for preenrichment.The majority of conventional methods used for detection and identification of pathogenic microorganisms, viruses, and/or their toxins lack the speed and sensitivity necessary for use in the field (they typically are not completed in a single day) and also require specialized equipment (20). Rapid methods, including antibody-based and nucleic acid-based assays, have revolutionized the methodology for detection of microbial pathogens and their toxins in foods (16). However, while most antibody-based and nucleic acid-based assays are rapid, specialized equipment is often required, and specific enrichment is needed to achieve the necessary sensitivity. This means that the analysis time can still be several days (16). Lateral-flow assays (LFAs) and column flow assays are tests that have considerable merit in terms of rapidity and ease of use in the field without specialized equipment (4, 5, 8, 19, 34).Two contrasting agents were used as detection targets in this study: (i) a potent microbial toxin (Clostridium botulinum neurotoxin), including type A, B, E, and F neurotoxins; and (ii) an infectious pathogen, Escherichia coli O157. These two targets present different problems for detection; the first target is a protein toxin, and the second target is intact bacterial cells. The botulinum neurotoxin is the most potent toxin known, and as little as 30 to 100 ng has the potential to be fatal to humans (28). It is responsible for botulism, a severe neuroparalytic disease that affects humans and also animals and birds (28). There are seven antigenically distinct botulinum neurotoxins (types A to G), and a number of subtypes have also been described (9, 11, 15, 28, 36). Botulism in humans is associated principally with neurotoxin types A, B, E, and F (27, 29). Since the botulinum neurotoxins are the toxic agents and they can be produced by six physiologically distinct clostridia (28), considerable emphasis has been placed on detection of the neurotoxins rather than the bacteria. The “gold standard” method for detecting botulinum neurotoxins is the mouse bioassay due to its high levels of sensitivity and specificity. However, this technique is also problematic (33). It typically requires 24 to 48 h to yield results, is expensive, and is becoming less favored because of its use of animals (4). The alternative tests include enzyme-linked immunosorbent assays (ELISAs), lateral-flow assays (LFAs), a chemiluminescent slot blot immunoassay, surface plasmon resonance (SPR), the assay with a large immunosorbent surface area (ALISSA) test, and quantum dot immunoassays (4, 5, 7, 22, 43, 46). Lateral-flow assays are available and are convenient for toxin testing as they are easy to perform and rapid (<30 min) and no additional equipment is required. However, their poor sensitivity has limited their use (23).E. coli O157 produces a cytotoxin (verotoxin), and an E. coli O157 infection can lead to severe bloody diarrhea, kidney failure, brain damage, and death. Enumeration, identification, and control of this pathogen are challenging due to the low infectious dose necessary to cause disease, which is between 2 and 2,000 ingested cells (41). Sources of E. coli O157 infection include ground beef and unpasteurized milk and apple juice (1), raw milk (6), and spinach and lettuce (42). Isolation of E. coli O157:H7 from water, food, and environmental samples is laborious. Culture is difficult due to the large competing microflora that either overgrows or mimics the non-sorbitol-fermenting organism E. coli O157:H7 (12). According to Tokarskyy and Marshall (41), the largest group of rapid test kits commercially available for testing for the presence of E. coli O157 in food includes immunological methods, such as latex agglutination, reverse passive latex agglutination, immunodiffusion, ELISA, immunomagnetic separation (IMS), and immunoprecipitation. The other methods that have been developed include a dipstick test device (2), a lateral-flow immunoassay (8), real-time PCR (39), and an enzyme-linked immunomagnetic chemiluminescent assay (17). However, in many cases these tests require preenrichment or have limited sensitivity.The objective of the work described here was to develop a rapid sensitive diagnostic test for detection of botulinum neurotoxins A, B, E, and F and E. coli O157 that can be used without preenrichment.     

Fates of Acid-Resistant and Non-Acid-Resistant Shiga Toxin-Producing Escherichia coli Strains in Ruminant Digestive Contents in the Absence and Presence of Probiotics

Healthy ruminants are the main reservoir of Shiga toxin-producing Escherichia coli (STEC). During their transit through the ruminant gastrointestinal tract, STEC encounters a number of acidic environments. As all STEC strains are not equally resistant to acidic conditions, the purpose of this study was to investigate whether acid resistance confers an ecological advantage to STEC strains in ruminant digestive contents and whether acid resistance mechanisms are induced in the rumen compartment. We found that acid-resistant STEC survived at higher rates during prolonged incubation in rumen fluid than acid-sensitive STEC and that they resisted the highly acidic conditions of the abomasum fluid, whereas acid-sensitive strains were killed. However, transit through the rumen contents allowed acid-sensitive strains to survive in the abomasum fluid at levels similar to those of acid-resistant STEC. The acid resistance status of the strains had little influence on STEC growth in jejunal and cecal contents. Supplementation with the probiotic Saccharomyces cerevisiae CNCM I-1077 or Lactobacillus acidophilus BT-1386 led to killing of all of the strains tested during prolonged incubation in the rumen contents, but it did not have any influence in the other digestive compartments. In addition, S. cerevisiae did not limit the induction of acid resistance in the rumen fluid. Our results indicate that the rumen compartment could be a relevant target for intervention strategies that could both limit STEC survival and eliminate induction of acid resistance mechanisms in order to decrease the number of viable STEC cells reaching the hindgut and thus STEC shedding and food contamination.Shiga toxin-producing Escherichia coli (STEC) strains are food-borne pathogens that cause human diseases ranging from uncomplicated diarrhea to hemorrhagic colitis (HC), as well as life-threatening complications, such as hemolytic-uremic syndrome (HUS). Most outbreaks and sporadic cases of HC and HUS have been attributed to O157:H7 STEC (http://www.cdc.gov/ecoli/outbreaks.html; http://www.euro.who.int). However, in some geographic areas, non-O157:H7 STEC infections are considered to be at least as important as E. coli O157:H7 infections, but they are often underdiagnosed (21, 46). In spite of diverse virulence characteristics, one common trait of pathogenic STEC strains could be resistance to the gastric acidity in humans. Indeed, it has been suggested that acid resistance of E. coli O157:H7 is negatively correlated with the infectious dose required for this organism to cause disease in humans (17).Healthy cattle and other ruminants appear to be the main reservoir of STEC strains. However, colonization of the cattle gastrointestinal tract (GIT) by STEC seems to be a transient event, with a mean duration of 14 days to 1 month (4, 8, 38). The site of STEC persistence and proliferation in the GIT depends on the STEC strain and seems to vary from one individual to another. Some previous studies identified the rumen as the primary site of colonization (8), whereas other studies referred to the cecum, the colon, or the rectum (10, 18, 23, 32, 42). Although STEC strains adhere in vitro to bovine colonic mucosa, forming the characteristic attaching and effacing lesions (35), they are very rarely associated with tissues in animal carriers and are generally isolated from the digesta (8). STEC does not, therefore, seem to colonize the gut mucosa, except for the anorectal mucosa, which has been described as the preferred colonization site for O157:H7 strains but not for non-O157:H7 strains (24, 32). During their transit through the ruminant GIT, STEC strains encounter various acidic conditions. Volatile fatty acid (VFA) concentrations are high in the rumen of grain-fed animals, and the pH may vary from 5.0 to 6.5. In these conditions, VFAs are in the undissociated form and can freely enter the bacterial cells, dissociate, and acidify the cytosol. In hay-fed animals, less fermentation occurs in the rumen, and the pH remains between 6.5 and 7. In the abomasum, STEC encounters strongly acidic conditions, regardless of the diet, due to the presence of mineral acids, resulting in a pH below 3. Then the pH increases from the proximal part to the distal part of the small intestine, and in the cecum and the colon STEC encounters more neutral pH conditions.All STEC strains are not equally resistant to acidic conditions (2, 9, 30, 45). Therefore, it could be hypothesized that acid-resistant (AR) STEC survives and persists better in the GIT of ruminants than acid-sensitive (AS) STEC. Acid resistance mechanisms can be induced during exposure to a moderately acidic environment (12, 26, 41). The rumen contents of a grain-fed animal could be such an environment favorable for the induction of acid resistance in STEC. While the diet does not seem to affect the acid resistance of an E. coli O157:H7 strain (19), grain feeding increases the number of acid-resistant generic coliforms (15, 19), either by inducing acid resistance mechanisms in the rumen or by selecting acid-resistant E. coli strains during passage through the abomasum. Hence, generic coliforms behave differently than E. coli O157:H7 in ruminants (19), and the potential ecological advantage conferred by acid resistance to non-O157:H7 STEC strains for persistence in the ruminant GIT has never been investigated.Inhibition of STEC proliferation in the ruminant gut may be mediated through probiotic supplementation. Several studies have demonstrated the capacity of certain lactic acid bacteria or yeast to reduce E. coli O157:H7 counts in vitro (1, 34) or in vivo (5, 40). The mechanisms of action of probiotics are not well characterized but could involve competition for nutrients and adhesion sites in the GIT, an increase in the VFA concentration and a decrease in the pH, production of antimicrobial molecules, or interference with quorum-sensing signaling (27-29). However, the impact of probiotics on non-O157:H7 STEC has been poorly investigated (36). Although not all non-O157:H7 STEC strains are pathogenic, limiting their carriage by ruminants should decrease the risk of food-borne illness. The impact of probiotics and of the physicochemical conditions of the rumen digesta on the survival of non-O157:H7 STEC strains or on induction of acid resistance mechanisms could have significant implications for farm management practices and food safety.The purpose of this work was to investigate whether the level of acid resistance, determined using an in vitro assay, confers an ecological advantage to STEC strains in ruminant digestive contents and whether acid resistance mechanisms are induced in the rumen compartment. Moreover, we evaluated the potential of probiotics to limit STEC survival and induction of acid resistance in the ruminant GIT.     

Longitudinal Study of Escherichia coli O157:H7 in a Beef Cattle Feedlot and Role of High-Level Shedders in Hide Contamination

The objectives of the study described here were (i) to investigate the dynamics of Escherichia coli O157:H7 fecal and hide prevalence over a 9-month period in a feedlot setting and (ii) to determine how animals shedding E. coli O157:H7 at high levels affect the prevalence and levels of E. coli O157:H7 on the hides of other animals in the same pen. Cattle (n = 319) were distributed in 10 adjacent pens, and fecal and hide levels of E. coli O157:H7 were monitored. When the fecal pen prevalence exceeded 20%, the hide pen prevalence was usually (25 of 27 pens) greater than 80%. Sixteen of 19 (84.2%) supershedder (>10⁴ CFU/g) pens had a fecal prevalence greater than 20%. Significant associations with hide and high-level hide (≥40 CFU/100 cm²) contamination were identified for (i) a fecal prevalence greater than 20%, (ii) the presence of one or more high-density shedders (≥200 CFU/g) in a pen, and (iii) the presence of one or more supershedders in a pen. The results presented here suggest that the E. coli O157:H7 fecal prevalence should be reduced below 20% and the levels of shedding should be kept below 200 CFU/g to minimize the contamination of cattle hides. Also, large and unpredictable fluctuations within and between pens in both fecal and hide prevalence of E. coli O157:H7 were detected and should be used as a guide when preharvest studies, particularly preharvest intervention studies, are designed.It is now well established that at the time of harvest, hides are the major source of Escherichia coli O157:H7 contamination on beef carcasses (1, 4, 22). Thus, reducing the levels of food-borne pathogens on cattle hides has been the focus of many pre- and postharvest research efforts. For postharvest applications, hide interventions (i.e., washing of hide-on carcasses with various antimicrobial agents) are direct approaches and have been shown to be efficacious for reducing hide and carcass contamination rates (2, 4, 5, 22).In the area of preharvest research, several approaches have been taken to reduce the prevalence of E. coli O157:H7 in feces of cattle presented for slaughter. These approaches include, among others, feeding cattle probiotics (dietary administration of beneficial bacteria to compete with E. coli O157:H7), vaccination, and bacteriophage treatment (8, 24, 30). These intervention approaches are indirect. By reducing the fecal pathogen load, the pathogen prevalence and the level on hides are reduced through lower cross-contamination at the feedlot, and subsequently, carcass contamination rates decrease. While the effectiveness of preharvest interventions varies, no preharvest intervention is 100% effective in reducing the fecal prevalence of E. coli O157:H7. It is not known what level of pathogen reduction in feces would be necessary to significantly reduce hide and carcass contamination during processing. Key pieces of information needed to address this question are the number of shedding cattle in a pen needed to contaminate the hides of most of the cattle in the same pen and at what level the shedding cattle are contaminated.Aside from the number of cattle shedding a pathogen, the concentration of the pathogen in feces plays a pivotal role in spreading the pathogen between animals. Recently, cattle shedding E. coli O157:H7 at levels of >10⁴ CFU/g (“supershedders”) have been associated with high rates of transmission of the pathogen between cohort animals (18, 23). Matthews et al. reported that 20% of the E. coli O157:H7 infections in cattle on Scottish farms were responsible for 80% of the transmission of the organism between animals (18). Another study reported similar findings; 9% of the animals shedding E. coli O157:H7 produced over 96% of the total E. coli O157:H7 fecal load for the group (23). While a number of studies have indicated the importance of supershedders in fecal transmission dynamics, there is a general lack of information concerning the effects of high shedding rates on hide prevalence and load. Accordingly, the objectives of this study were (i) to investigate the dynamics of E. coli O157:H7 prevalence and levels in feces and on hides of feedlot cattle over time and (ii) to determine how pathogen prevalence and levels on hides in a pen are affected by individuals shedding E. coli O157:H7 at high levels.In the analysis presented here, fecal shedding was analyzed using the following three categories based on the level of E. coli O157:H7 being shed: shedding positive (presumed concentration, ≥1 CFU/g), high-density shedder (≥200 CFU/g), and supershedder (≥10⁴ CFU/g). Several definitions of E. coli O157:H7 supershedders have been offered previously. One-time shedding levels of >10³ or >10⁴ CFU/g have been used in multiple studies (17, 23, 24), while other groups have required persistent colonization of the rectoanal junction, as well as high cell counts, for an animal to qualify as a supershedder (10). Recently, Chase-Topping et al. (9) reviewed the requirements for supershedder status and provided a working definition: an animal that excretes >10⁴ CFU/g. In doing this, Chase-Topping et al. noted the high stringency of this definition and acknowledged that with such a definition some supershedders will be missed if they are sampled at times other than peak shedding times (9). In the current study, this was a concern. In an attempt to investigate the link between high-shedding-level animals and hide contamination, greater leeway was needed in the classification. When it is sampled on a monthly basis, an animal shedding at high levels can have a large impact on the hide status of pen cohorts between sampling intervals but not be shedding at peak levels on the day of sample collection. Hence, the categories described above were selected to analyze the relationship between fecal shedding and hide contamination.     

Potential Applicability of Chymotrypsin-Susceptible Microcin J25 Derivatives to Food Preservation

Microcin J25 (MccJ25) is a 21-residue ribosomally synthesized lariat peptide antibiotic. MccJ25 is active against such food-borne disease-causing pathogens as Salmonella spp., Shigella spp., and Escherichia coli, including E. coli O157:H7 and non-O157 strains. MccJ25 is highly resistant to digestion by proteolytic enzymes present in the stomach and intestinal contents. MccJ25 would therefore remain active in the gastrointestinal tract, affecting normal intestinal microbiota, and this limits the potential use of MccJ25 as a food preservative. In the present paper, we describe a chymotrypsin-susceptible MccJ25 derivative with a mutation of Gly¹² to Tyr that retained almost full antibiotic activity and efficiently inhibited the growth of pathogenic Salmonella enterica serovar Newport and Escherichia coli O157:H7 in skim milk and egg yolk. However, unlike the wild-type MccJ25, the MccJ25(G12Y) variant was inactivated by digestive enzymes both in vitro and in vivo. To our knowledge, our results represent the first example of a rational modification of a microcin aimed at increasing its potential use in food preservation.Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide consisting of 21 amino acid residues (G¹-G-A-G-H⁵-V-P-E-Y-F¹⁰-V-G-I-G-T¹⁵-P-I-S-F-Y²⁰-G) (4, 12). Four genes (mcjA, mcjB, mcjC, and mcjD) are required for MccJ25 synthesis, export, and immunity (14, 15). The mcjA gene encodes a 58-amino-acid MccJ25 precursor, which is processed by the products of mcjC and mcjB (7). Once synthesized, the mature MccJ25 is excreted to the medium by McjD, an ABC-type transporter (6, 14). The tertiary structure of MccJ25 was elucidated as a lariat peptide (1, 10, 17). It contains an eight-residue ring (G¹ to E⁸) and a tail (Y⁹ to G²¹) whose C-terminal end is sterically trapped within the ring. MccJ25 amino acids F¹⁰ to P¹⁶ form a β-hairpin structure, comprising two β-strands (F¹⁰-V¹¹ and T¹⁵-P¹⁶) and a β-turn (V¹¹ to G¹⁴).MccJ25 is active on gram-negative bacteria related to the producer strain, and among them are several human pathogens (11, 12, 16). It was previously shown that the E. coli RNA polymerase (5, 18) and the bacterial respiratory chain (2, 9) are the targets for MccJ25 action. MccJ25 is active on pathogenic strains of Salmonella spp., Shigella spp. (12), and E. coli, including O157:H7 (11) as well as non-O157 strains (data not shown), that frequently cause outbreaks of food-borne diseases. In addition, Sable et al. (11) showed that MccJ25 was the most active microcin against 12 out of 15 diarrheagenic E. coli strains tested. These authors also demonstrated that MccJ25 inhibits E. coli O157:H7 in biological products such as milk, egg yolk, and meat extract. These findings suggest that MccJ25 could be an efficient complement to nisin for food preservation. However, the potential usefulness of MccJ25 is compromised by the fact that it is highly resistant to digestion by proteolytic enzymes of the stomach (pepsin) and intestinal (trypsin, chymotrypsin, and carboxypeptidases) contents. Thus, the antibiotic would most likely remain active in the intestine, and this could lead to disturbance of the normal microbiota. Therefore, for potential use of MccJ25 as a food additive, it would be desirable to render MccJ25 susceptible to at least one of these proteases. In the present work, we describe a chymotrypsin-susceptible MccJ25 derivative that remains fully active on S. Newport and E. coli O157:H7 in biological products, namely milk and egg yolk. In addition, we demonstrate that the peptide is inactivated by rat intestinal contents.