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).     

Escherichia coli O157:H7 Strains That Persist in Feedlot Cattle Are Genetically Related and Demonstrate an Enhanced Ability To Adhere to Intestinal Epithelial Cells

A longitudinal study was conducted to investigate the nature of Escherichia coli O157:H7 colonization of feedlot cattle over the final 100 to 110 days of finishing. Rectal fecal grab samples were collected from an initial sample population of 788 steers every 20 to 22 days and microbiologically analyzed to detect E. coli O157:H7. The identities of presumptive colonies were confirmed using a multiplex PCR assay that screened for gene fragments unique to E. coli O157:H7 (rfbE and fliC_h7) and other key virulence genes (eae, stx₁, and stx₂). Animals were classified as having persistent shedding (PS), transient shedding (TS), or nonshedding (NS) status if they consecutively shed the same E. coli O157:H7 genotype (based on the multiplex PCR profile), exhibited variable E. coli O157 shedding, or never shed morphologically typical E. coli O157, respectively. Overall, 1.0% and 1.4% of steers were classified as PS and NS animals, respectively. Characterization of 132 E. coli O157:H7 isolates from PS and TS animals by pulsed-field gel electrophoresis (PFGE) typing yielded 32 unique PFGE types. One predominant PFGE type accounted for 53% of all isolates characterized and persisted in cattle throughout the study. Isolates belonging to this predominant and persistent PFGE type demonstrated an enhanced (P < 0.0001) ability to adhere to Caco-2 human intestinal epithelial cells compared to isolates belonging to less common PFGE types but exhibited equal virulence expression. Interestingly, the attachment efficacy decreased as the genetic divergence from the predominant and persistent subtype increased. Our data support the hypothesis that certain E. coli O157:H7 strains persist in feedlot cattle, which may be partially explained by an enhanced ability to colonize the intestinal epithelium.Escherichia coli serotype O157:H7 was first linked to human illness in the early 1980s, when it was determined to cause severe abdominal pain with initially watery diarrhea that progressed to grossly bloody diarrhea accompanied by little or no fever (42). Initially, E. coli O157:H7 can cause nonbloody diarrhea through attachment to, and subsequent destruction of, intestinal microvilli (24). In addition to microvillus damage, serious health complications can arise due to the ability of E. coli O157:H7 to produce Shiga toxins (Stx1 and Stx2). Shiga toxins are very potent cytotoxins that are absorbed into the intestinal microvasculature and initiate apoptosis of vascular epithelium, resulting in hemorrhagic colitis (41). Persistent uptake of these toxins may lead to more severe manifestations of disease, such as hemolytic-uremic syndrome, which may ultimately result in kidney failure (24). Most recent estimates have identified E. coli O157:H7 as the cause of at least 70,000 cases of food-borne illness annually in the United States, and in 4% of cases life-threatening hemolytic-uremic syndrome develops (37). Epidemiological studies have implicated the consumption of meat, dairy products, produce, and water contaminated by animal feces, as well as person-to-person contact and direct contact with farm animals or their environment, as routes of E. coli O157:H7 transmission leading to human illness (36).It is generally accepted that cattle and other animals are the major reservoir of E. coli O157:H7, but it is still not clear if animals are colonized for prolonged periods with E. coli O157:H7 or if they transiently shed this organism following repeated exposure to it through ingestion of contaminated feedstuffs or water or through exposure to other contaminated environmental sources. Based on results of numerous epidemiological studies (4, 6, 21, 30, 32), the prevalence of E. coli O157:H7 in feedlot cattle is highly variable and can range from less than 1% to 80%. Several other studies (7, 8, 23) have found evidence of persistent E. coli O157:H7 colonization in individual cattle, supporting the hypothesis that at least some animals are susceptible to persistent E. coli O157:H7 colonization. Multiple experimental inoculation studies (15, 23, 39, 46) showed that E. coli O157:H7 persists in the bovine gastrointestinal (GI) tract for at least 14 days up to 140 days postinfection. Studies have implicated the lower GI tract and specifically the recto-anal junction (RAJ) as the major location of E. coli O157:H7 colonization and proliferation (9, 12, 23, 39); however, this organism also can be found throughout the bovine GI tract (7, 8, 31, 40, 54).It stands to reason that if the E. coli O157:H7 prevalence in cattle presented for harvest were reduced, there would be a decrease in the probability of beef product contamination, if good manufacturing procedures were used. Although there is consensus concerning the importance of preharvest pathogen mitigation and its role in minimizing entry of E. coli O157:H7 into harvest facilities, there is disagreement about the significance of “supershedders” (animals that excrete large quantities of a pathogen for various amounts of time) for E. coli O157:H7 transmission dynamics at the preharvest level (12, 34, 35, 39). Utilizing statistical modeling, researchers have estimated that, on average, the prevalence of “supershedders” in a population is 4% and that these animals excrete 50 times more E. coli O157:H7 than other animals colonized by this organism (34). Additionally, the same researchers suggested that approximately 80% of E. coli O157:H7 transmission is generated by a few “supershedders” (35).Research by our group discovered a unique association between E. coli O157:H7 prevalence in pen floor fecal pats and carcass contamination by this pathogen (57). When the prevalence in fecal pats from a pen floor exceeded 20%, carcasses of animals from the pen had E. coli O157:H7 prevalence values of 14.3, 2.9, and 0.7% before evisceration, after evisceration, and after final intervention, respectively. However, when the prevalence in pen floor fecal pats was less than 20%, the preeviscerated carcass prevalence value was 6.3%, and there was no detectable E. coli O157:H7 contamination of carcass samples after evisceration and after final intervention (57). Thus, we hypothesize that animals which persistently excrete normal levels of E. coli O157:H7 over prolonged periods (persistent shedders [PS]) rather than animals that periodically shed abnormally high levels (supershedders) are the most significant source of E. coli O157:H7 contamination in the food continuum. Although previous studies suggested that cattle may be persistently colonized by E. coli O157:H7 and shed this organism in their feces for prolonged periods, molecular subtyping data are required to further investigate whether cattle are persistently colonized by the same strain (i.e., molecular subtype) or if they are repeatedly exposed to different strains through contaminated feedstuffs, water, or other environmental sources. Thus, the objectives of this study were to determine if naturally colonized feedlot cattle persistently shed E. coli O157:H7, using combined cultural microbiological analyses, molecular subtyping approaches, and in vitro virulence phenotype assays to probe the factors (agent, host, environment, or a combination of these factors) that contribute to the complex ecology of E. coli O157:H7 persistence at the preharvest level.     

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.     

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.     

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.     

Increased Antibiotic Resistance of Escherichia coli in Mature Biofilms

Biofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility of Escherichia coli cells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 μg/ml), kanamycin (25 μg/ml), and ofloxacin (10 μg/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced by rpoS expression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction of rpoS-mediated stress responses.Reduced susceptibility of biofilm bacteria to antimicrobial agents is a crucial problem for treatment of chronic infections (11, 29, 48). It has been estimated that 65% of microbial infections are associated with biofilms (11, 29, 37), and biofilm cells are 100 to 1,000 times more resistant to antimicrobial agents than planktonic bacterial cells (11, 29, 32).The molecular nature of this apparent resistance has not been elucidated well, and a number of mechanisms have been proposed to explain the reduced susceptibility, such as restricted antibiotic penetration (47), decreased growth rates and metabolism (7, 52), quorum sensing and induction of a biofilm-specific phenotype (8, 29, 35, 39, 49), stress response activation (7, 52), and an increase in expression of efflux pumps (14). Biofilm resistance has generally been assumed to be due to the fact that the cells in the deeper layers of thick biofilms, which grow more slowly, have less access to antibiotics and nutrients. However, this is not the only reason in many cases. Familiar mechanisms of antibiotic resistance, such as modifying enzymes and target mutations, do not seem to be responsible for the biofilm resistance. Even sensitive bacteria that do not have a known genetic basis for resistance can exhibit profoundly reduced susceptibility when they form biofilms (48).It was reported previously that changes in gene expression induced a biofilm-specific phenotype (5, 13, 22, 35, 41, 42). Several genes have been proposed to be particularly important for biofilm formation, and the importance of the rpoS gene in Escherichia coli biofilm formation was suggested recently (1, 10, 22, 42). It has been suggested that induction of an rpoS-mediated stress response results in physiological changes that could contribute to antibiotic resistance (29). Although several mechanisms and genes have been proposed to explain biofilm resistance to antibiotics, this resistance is not still fully understood because these mechanisms seem to work together within a biofilm community. In addition, the physiology of biofilm cells is remarkably heterogeneous and varies according to the location of individual cells within biofilms (33, 34, 46).In this study, susceptibility of E. coli cells in biofilms to antibiotics was investigated. The E. coli cells in the deeper layers of mature biofilms were directly treated with three antibiotics with different molecular targets, the β-lactam ampicillin, the aminoglycoside kanamycin, and the fluoroquinolone ofloxacin. The biofilm biomass was removed before antibiotic treatment, and only the cells located in the deeper layers of the mature biofilms were directly exposed to antibiotics; thus, the effects of restricted antibiotic and nutrient penetration, as well as heterogeneous physiological states in biofilms, were reduced. Although ofloxacin and kanamycin effectively killed the biofilm cells, ampicillin could not kill the cells, which led to regrowth of biofilms. However, the cells in young colony biofilms were completely killed by ampicillin. Therefore, to determine which genes are induced in the mature biofilm cells, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. Based on the experimental data obtained, possible mechanisms of the increased biofilm resistance to ampicillin are discussed below.     

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.     

Conjugative Plasmid Transfer and Adhesion Dynamics in an Escherichia coli Biofilm

A conjugative plasmid from the catheter-associated urinary tract infection strain Escherichia coli MS2027 was sequenced and annotated. This 42,644-bp plasmid, designated pMAS2027, contains 58 putative genes and is most closely related to plasmids belonging to incompatibility group X (IncX1). Plasmid pMAS2027 encodes two important virulence factors: type 3 fimbriae and a type IV secretion (T4S) system. Type 3 fimbriae, recently found to be functionally expressed in E. coli, played an important role in biofilm formation. Biofilm formation by E. coli MS2027 was specifically due to expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027 and enabled a non-biofilm-forming strain to grow as part of a mixed biofilm following acquisition of this plasmid. Thus, the importance of conjugation as a mechanism to spread biofilm determinants was demonstrated. Conjugation may represent an important mechanism by which type 3 fimbria genes are transferred among the Enterobacteriaceae that cause device-related infections in nosocomial settings.Bacterial biofilms are complex communities of bacterial cells living in close association with a surface (17). Bacterial cells in these protected environments are often resistant to multiple factors, including antimicrobials, changes in the pH, oxygen radicals, and host immune defenses (19, 38). Biofilm formation is a property of many bacterial species, and a range of molecular mechanisms that facilitate this process have been described (2, 3, 11, 14, 16, 29, 33, 34). Often, the ability to form a biofilm is dependent on the production of adhesins on the bacterial cell surface. In Escherichia coli, biofilm formation is enhanced by the production of certain types of fimbriae (e.g., type 1 fimbriae, type 3 fimbriae, F1C, F9, curli, and conjugative pili) (14, 23, 25, 29, 33, 39, 46), cell surface adhesins (e.g., autotransporter proteins such as antigen 43, AidA, TibA, EhaA, and UpaG) (21, 34, 35, 40, 43), and flagella (22, 45).The close proximity of bacterial cells in biofilms creates an environment conducive for the exchange of genetic material. Indeed, plasmid-mediated conjugation in monospecific and mixed E. coli biofilms has been demonstrated (6, 18, 24, 31). The F plasmid represents the best-characterized conjugative system for biofilm formation by E. coli. The F pilus mediates adhesion to abiotic surfaces and stabilizes the biofilm structure through cell-cell interactions (16, 30). Many other conjugative plasmids also contribute directly to biofilm formation upon derepression of the conjugative function (16).One example of a conjugative system employed by gram-negative Enterobacteriaceae is the type 4 secretion (T4S) system. The T4S system is a multisubunit structure that spans the cell envelope and contains a secretion channel often linked to a pilus or other surface filament or protein (8). The Agrobacterium tumefaciens VirB-VirD4 system is the archetypical T4S system and is encoded by 11 genes in the virB operon and one gene (virD4) in the virD operon (7, 8). Genes with strong homology to genes in the virB operon have also been identified on other conjugative plasmids. For example, the pilX1 to pilX11 genes on the E. coli R6K IncX plasmid and the virB1 to virB11 genes are highly conserved at the nucleotide level (28).We recently described identification and characterization of the mrk genes encoding type 3 fimbriae in a uropathogenic strain of E. coli isolated from a patient with a nosocomial catheter-associated urinary tract infection (CAUTI) (29). The mrk genes were located on a conjugative plasmid (pMAS2027) and were strongly associated with biofilm formation. In this study we determined the entire sequence of plasmid pMAS2027 and revealed the presence of conjugative transfer genes homologous to the pilX1 to pilX11 genes of E. coli R6K (in addition to the mrk genes). We show here that biofilm formation is driven primarily by type 3 fimbriae and that the T4S apparatus is unable to mediate biofilm growth in the absence of the mrk genes. Finally, we demonstrate that conjugative transfer of pMAS2027 within a mixed biofilm confers biofilm formation properties on recipient cells due to acquisition of the type 3 fimbria-encoding mrk genes.     

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.     

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).     

New Device for High-Throughput Viability Screening of Flow Biofilms

Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well (“static”) biofilms are available, there are no methods for such screening of continuous flow biofilms (“flow biofilms”). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of Pseudomonas aeruginosa PAO1 flow biofilms.Bacterial biofilms are surface-attached communities that are encased in a polymeric matrix, which exhibit a high degree of resistance to antimicrobial agents and the host immune system (12, 16). This makes them medically important; diseases with a biofilm component are chronic and difficult to eradicate. Examples of such diseases are cystitis (1), endocarditis (31), cystic fibrosis (35), and middle-ear (17) and indwelling medical device-associated (20) infections. Biofilms also play important environmental roles in, for example, wastewater treatment (38), bioremediation (29, 30), biofouling (7), and biocorrosion (2). Better control of biofilms requires elucidation of the molecular basis of their superior resistance (by identifying resistance-compromised mutants) and identification of compounds with antibiofilm activity. While our understanding of these aspects of biofilms has increased (11, 15, 25-27, 36), further work, including development of accurate high-throughput (HTP) methods for screening biofilm viability, is needed.Two major biofilm models are studied in the laboratory, biofilms grown without a continuous flow of fresh medium and biofilms grown with a continuous flow of fresh medium; examples of these two models are microtiter well biofilms and flow cell biofilms, respectively. Methods have been developed for HTP screening of the viability of static biofilms (6, 28, 32, 33), but there are no methods for HTP screening of flow biofilms. The latter biofilms are typically grown in flow cells, which have to be examined individually to determine viability and thus cannot be used for rapid screening. An HTP screening method for flow biofilms is desirable, as these biofilms more closely approximate natural biofilms and can differ from static biofilms evidently due to hydrodynamic influences on cell signaling (22, 34). For example, the ability of rpoS-deficient Escherichia coli (lacking σ^S) to form flow biofilms is impaired, but its capacity to form biofilms under static conditions is enhanced (18).We describe here a new application of a recently developed device (8-10, 13), the “BioFlux” device consisting of microfluidic channels for biofilm growth. Other microfluidic devices have recently been used for biofilm formation (14, 19, 21, 23), but none of them has been used for HTP screening. The BioFlux device permits rapid measurement of the fluorescence of flow biofilms with a plate reader, which permits initial HTP screening of the viability of such biofilms.