Dianna M. Jordan

Avian-Pathogenic Escherichia coli Strains Are Similar to Neonatal Meningitis E. coli Strains and Are Able To Cause Meningitis in the Rat Model of Human Disease

ABSTRACT Escherichia coli strains causing avian colibacillosis and human neonatal meningitis, urinary tract infections, and septicemia are collectively known as extraintestinal pathogenic E. coli (ExPEC). Characterization of ExPEC strains using various typing techniques has shown that they harbor many similarities, despite their isolation from different host species, leading to the hypothesis that ExPEC may have zoonotic potential. The present study examined a subset of ExPEC strains: neonatal meningitis E. coli (NMEC) strains and avian-pathogenic E. coli (APEC) strains belonging to the O18 serogroup. The study found that they were not easily differentiated on the basis of multilocus sequence typing, phylogenetic typing, or carriage of large virulence plasmids. Among the APEC strains examined, one strain was found to be an outlier, based on the results of these typing methods, and demonstrated reduced virulence in murine and avian pathogenicity models. Some of the APEC strains tested in a rat model of human neonatal meningitis were able to cause meningitis, demonstrating APECs ability to cause disease in mammals, lending support to the hypothesis that APEC strains have zoonotic potential. In addition, some NMEC strains were able to cause avian colisepticemia, providing further support for this hypothesis. However, not all of the NMEC and APEC strains tested were able to cause disease in avian and murine hosts, despite the apparent similarities in their known virulence attributes. Thus, it appears that a subset of NMEC and APEC strains harbors zoonotic potential, while other strains do not, suggesting that unknown mechanisms underlie host specificity in some ExPEC strains.

Intestinal adherence associated with type IV pili of enterohemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) by colonizing the gut mucosa and producing Shiga toxins (Stx). The only factor clearly demonstrated to play a role in EHEC adherence to intestinal epithelial cells is intimin, which binds host cell integrins and nucleolin, as well as a receptor (Tir) that it injects into the host cell. Here we report that EHEC O157:H7 produces adhesive type IV pili, which we term hemorrhagic coli pilus (HCP), composed of a 19-kDa pilin subunit (HcpA) that is encoded by the hcpA chromosomal gene. HCP were observed as bundles of fibers greater than 10 microm in length that formed physical bridges between bacteria adhering to human and bovine host cells. Sera of HUS patients, but not healthy individuals, recognized HcpA, suggesting that the pili are produced in vivo during EHEC infections. Inactivation of the hcpA gene in EHEC EDL933 resulted in significantly reduced adherence to cultured human intestinal and bovine renal epithelial cells and to porcine and bovine gut explants. An escN mutant, which is unable to translocate Tir, adhered less than the hcpA mutant, suggesting that adherence mediated by intimin-Tir interactions is a prelude to HCP-mediated adherence. An hcpA and stx1,2 triple mutant and an hcpA mutant had similar levels of adherence to bovine and human epithelial cells while a stx1,2 double mutant had only a minor defect in adherence, indicating that HCP-mediated adherence and cytotoxicity are independent events. Our data establish that EHEC O157:H7 HCP are intestinal colonization factors that are likely to contribute to the pathogenic potential of this food-borne pathogen.

Long Polar Fimbriae Contribute to Colonization by Escherichia coli O157:H7 In Vivo

ABSTRACT The contribution of long polar fimbriae to intestinal colonization by Escherichia coli O157:H7 was evaluated in sheep, conventional pigs, and gnotobiotic piglets. E. coli O157:H7 strains with lpfA1 and lpfA2 mutated were recovered in significantly lower numbers and caused fewer attachment and effacement lesions than the parent strain.

Emergence of a Tetracycline-Resistant Campylobacter jejuni Clone Associated with Outbreaks of Ovine Abortion in the United States

ABSTRACT Campylobacter infection is one of the major causes of ovine abortions worldwide. Historically, Campylobacter fetus subsp. fetus was the major cause of Campylobacter-associated abortion in sheep; however, Campylobacter jejuni is increasingly associated with sheep abortions. We examined the species distribution, genotypes, and antimicrobial susceptibilities of abortion-associated Campylobacter isolates obtained from multiple lambing seasons on different farms in Iowa, Idaho, South Dakota, and California. We found that C. jejuni has replaced C. fetus as the predominant Campylobacter species causing sheep abortion in the United States. Most strikingly, the vast majority (66 of 71) of the C. jejuni isolates associated with sheep abortion belong to a single genetic clone, as determined by pulsed-field gel electrophoresis, multilocus sequence typing, and cmp gene (encoding the major outer membrane protein) sequence typing. The in vitro antimicrobial susceptibilities of these isolates to the antibiotics that are routinely used in food animal production were determined using the agar dilution test. All of the 74 isolates were susceptible to tilmicosin, florfenicol, tulathromycin, and enrofloxacin, and 97% were sensitive to tylosin. However, all were resistant to tetracyclines, the only antibiotics currently approved in the United States for the treatment of Campylobacter abortion in sheep. This finding suggests that feeding tetracycline for the prevention of Campylobacter abortions is ineffective and that other antibiotics should be used for the treatment of sheep abortions in the United States. Together, these results indicate that a single tetracycline-resistant C. jejuni clone has emerged as the major cause of Campylobacter-associated sheep abortion in the United States.

Sequence Analysis and Characterization of a Transferable Hybrid Plasmid Encoding Multidrug Resistance and Enabling Zoonotic Potential for Extraintestinal Escherichia coli

ABSTRACT ColV plasmids of extraintestinal pathogenic Escherichia coli (ExPEC) encode a variety of fitness and virulence factors and have long been associated with septicemia and avian colibacillosis. These plasmids are found significantly more often in ExPEC, including ExPEC associated with human neonatal meningitis and avian colibacillosis, than in commensal E. coli. Here we describe pAPEC-O103-ColBM, a hybrid RepFIIA/FIB plasmid harboring components of the ColV pathogenicity island and a multidrug resistance (MDR)-encoding island. This plasmid is mobilizable and confers the ability to cause septicemia in chickens, the ability to cause bacteremia resulting in meningitis in the rat model of human disease, and the ability to resist the killing effects of multiple antimicrobial agents and human serum. The results of a sequence analysis of this and other ColV plasmids supported previous findings which indicated that these plasmid types arose from a RepFIIA/FIB plasmid backbone on multiple occasions. Comparisons of pAPEC-O103-ColBM with other sequenced ColV and ColBM plasmids indicated that there is a core repertoire of virulence genes that might contribute to the ability of some ExPEC strains to cause high-level bacteremia and meningitis in a rat model. Examination of a neonatal meningitis E. coli (NMEC) population revealed that approximately 58% of the isolates examined harbored ColV-type plasmids and that 26% of these plasmids had genetic contents similar to that of pAPEC-O103-ColBM. The linkage of the ability to confer MDR and the ability contribute to multiple forms of human and animal disease on a single plasmid presents further challenges for preventing and treating ExPEC infections.

The Escherichia coli ycbQRST operon encodes fimbriae with laminin‐binding and epithelial cell adherence properties in Shiga‐toxigenic E. coli O157:H7

The human pathogen Shiga-toxigenic Escherichia coli (STEC) O157:H7 contains a ycbQRST fimbrial-like operon, which shares significant homology to the family of F17 fimbrial biogenesis genes f17ADCG found in enterotoxigenic E.  coli. We report that growth of STEC O157:H7 strain EDL933 in minimal Minca medium at 37°C and during adherence to epithelial cells led to the production of fine peritrichous fimbriae, which were found to be composed of a major subunit of 18  kDa whose N-terminal amino acid sequence matched the predicted protein product of the ycbQ gene; and showed significant homology to the F17a-A fimbrin. Similar to the F17 fimbriae, the purified STEC fimbriae and the recombinant YcbQ protein fused to a His peptide tag bound laminin, but not fibronectin or collagen. Thus, we propose the name E.  coli YcbQ laminin-binding fimbriae (ELF) to designate the fimbriae encoded by the ycbQRST operon. The role of ELF as an adherence factor of STEC to cultured epithelial cells was investigated. We provide compelling evidence demonstrating that ELF contributes to adherence of STEC to human intestinal epithelial cells and to cow and pig gut tissue in vitro. Deletion in the fimbrin subunit gene elfA (or ycbQ) in STEC strain EDL933 led to an isogenic strain, which showed significant reduction (60%) in adherence to HEp-2 cells in comparison with the parental strain. In addition, antibodies against the purified ELF also partially blocked adherence of two STEC O157:H7 strains. These observations suggest that ELF functions as an accessory adherence factor that, along with other known redundant adhesins, contributes to the overall adhesive properties of STEC O157:H7 providing these organisms with ecological advantages to survive in different hosts and in the environment.

Detection of Lawsonia Intracellularis in Swine using Polymerase Chain Reaction Methodology

The polymerase chain reaction (PCR) was evaluated for its usefulness as a diagnostic tool to detect Lawsonia (ileal symbiont) intracellularis. Porcine ilea were collected from swine cases submitted to the Iowa State University Veterinary Diagnostic Laboratory between December 1, 1994, and June 30, 1995. Sampling was random, with no regard to health status. There were 621 ileum scrapings evaluated using the PCR technique. Thirty-five of the samples were positive, either by PCR or conventional diagnostic methods such as histology and Warthin-Starry silver stain. These 35 samples were further evaluated by Warthin-Starry silver stain and indirect immunofluorescent antibody test (IFAT) to confirm the presence of L. intracellularis in the tissue sections. Of the 26 samples positive by PCR, 22 were positive by IFAT. Sixteen of the 22 were also positive when stained with Warthin-Starry and evaluated microscopically for typical bacteria. Nine of the original samples were negative by all 3 techniques. PCR appears more sensitive and specific for L. intracellularis detection than Warthin-Starry stain and IFAT. This study provides evidence that PCR may be useful as a reference standard for the detection of L. intracellularis. PCR may be an appropriate monitoring tool for swine herds because it is a rapid procedure that could be applied to batch testing. Although the test is currently too laborious and expensive for routine diagnostic use, there may be situations in which it is justified because of the advantages of greater sensitivity and specificity.

Escherichia coli O157:H7 does not require intimin to persist in pigs.

Pigs dually inoculated with Escherichia coli O157:H7 and an isogenic intimin deletion mutant were examined 1 h to 38 days postinoculation. Both strains were recovered from the alimentary tract without sustained significant differences between strains. The highest levels were recovered from the spiral colon early postinoculation and from the tonsil later in the study. There is evidence that intimin facilitates the persistence of Escherichia coli O157:H7 in adult ruminants (6). In cattle and sheep, E. coli O157:H7 strain 86-24 was recovered in higher numbers from feces 2 weeks and 1 month after experimental inoculation than was the isogenic intimin-deficient mutant. Strain 86-24 was present in seven out of eight sheep and calves, whereas only one sheep and one calf remained infected with the intimin-deficient mutant at 1 month postinfection. Intimin is necessary for the development of attachment-and-effacement lesions in E. coli O157:H7-infected newborn pigs (9, 11, 17, 22). It has been demonstrated that older pigs can be infected by E. coli O157:H7 and maintain the infection for at least 2 months with recovery of the inoculum E. coli O157:H7 from the feces (4). There have been no studies to determine what, if any, role intimin plays in the persistence of E. coli O157:H7 in such older pigs. Since pork can serve as a vehicle for food-borne pathogens (1, 12, 13, 24), it is important to define the factors that influence persistent colonization of E. coli O157:H7 in pigs. The objective of this study was to determine if intimin facilitates persistence of E. coli O157:H7 in older pigs. Ten-week-old conventional pigs were acclimated for 2 weeks to the facilities and antibiotic-free feed; preinoculation fecal samples were collected. The bacterial inoculum strains were E. coli O157:H7 strain 86-24, resistant to nalidixic acid (6, 15), and an intimin-deficient strain, E. coli O157:H7 86-24 Δeae10, that was resistant to streptomycin (6, 17). Bacterial strains were grown as previously described, and inoculum cultures were harvested, quantified, and frozen at −80°C until needed (4, 5, 20). The inoculum contained both the parent strain and the intimin mutant strain at a dose of 1010 CFU per strain. The inoculum was given via the feed to 49 pigs. Two pigs served as noninoculated controls. Four to six pigs were necropsied at scheduled intervals postinoculation (1, 6, 12, 24, and 48 h and 4, 12, 24, 31, and 38 days). Tissue samples from the soft palate adjacent to the tonsil, the tonsil, the stomach, the cecum, the spiral colon, and the rectal-anal junction and fecal samples were collected for bacteriologic analysis. The preinoculation fecal samples were processed and plated onto MacConkey agar, sorbitol MacConkey agar, and sorbitol MacConkey agar with streptomycin-novobiocin and nalidixic acid-novobiocin antibiotic combinations added to suppress E. coli strains other than the inoculum strains. Fecal and tissue samples were collected and processed for bacteriologic evaluation as previously described (3-7). Tonsil, cecum, and rectal-anal junction tissue samples were collected at necropsy and processed for histologic examination. Slides were stained with hematoxylin and eosin, and another set was stained for E. coli O157 antigen (8). Selected sections from the paraffin blocks were prepared for transmission electron microscopy by staining with 1% osmium tetroxide, cut into thin sections, and placed on nickel grids for viewing with a Philips 410 transmission electron microscope. The results were analyzed as correlated observations. Distribution of differences was used for statistical evaluation. The intimin-deficient strain count was subtracted from the parent strain count, resulting in a sample difference for each sample. If samples were only positive on enrichment, an arbitrary number of 49 was assigned for statistical analysis. The differences were averaged at each time point, and the standard deviation and probability interval were calculated. Each set of data was evaluated to determine if zero was within the probability interval. JMP (version 5.0.1a; SAS Institute, Inc.) was used for analysis, with a P value of <0.05 indicating a significant difference. Both inoculum strains of E. coli O157:H7 were recovered from all inoculated pigs, with little difference between strains, as determinated by bacterial counts or the number of tissues positive throughout the study (Table ​(Table1).1). The tonsil, soft palate, cecum, and spiral colon had recoverable levels of one or both inoculum strains throughout the study, with bacterial counts tending to be highest in the spiral colon (Fig. ​(Fig.1).1). The parent strain was isolated in a statistically significantly higher quantity than the intimin-deficient strain in six instances: from the rectal-anal junction at 6 h postinoculation, from the cecum at 24 and 48 h postinoculation, and from the spiral colon, rectal-anal junction, and feces at 48 h postinoculation. The parent strain was not isolated consistently more frequently or in greater quantity from tissue samples than was the intimin-deficient mutant strain (Table ​(Table1;1; Fig. ​Fig.11). FIG. 1. Bacterial isolation from tissues and feces. Shown are individual log10 bacterial counts of the E. coli O157:H7 parent strain (squares) and its intimin-deficient mutant (triangles) obtained by direct plating and enrichment of tissue and fecal samples from ... TABLE 1. Tissue and fecal recovery of inoculum strainsa Histologically, there were low numbers of bacteria intimately associated with the mucosa of cecal sections. Three of the cecal samples contained O157-antigen-positive organisms closely associated with enterocytes. The antigen-positive organisms could have been either of the inoculum strains. In some instances, the enterocyte brush border was interrupted and the cells were somewhat attenuated; however, evidence of effacement was not compelling. All three of these samples were from pigs necropsied at 6 h postinoculation. Sections from these samples were also examined by electron microscopy; no attaching-and-effacing lesions were observed. There have been many other reports of studies of E. coli O157:H7 infection and persistence in ruminants but few reports of studies of nonruminant species (2, 4-7, 14, 16, 18, 23). The inability to demonstrate reduced persistence by the intimin-deficient mutant strain in pigs contrasts with similar studies of these strains in cattle and sheep (6). In aggregate, the studies lead to the suggestion that intimin contributes more to persistence in ruminants than in swine. The complete mechanism of colonization by E. coli O157:H7 is unclear. Other Shiga-toxigenic E. coli strains lacking intimin presumably have other adhesions facilitating colonization and pathogenicity (10, 16, 19, 21). These or similar factors are likely active or present in strains possessing intimin as well. It is likely that unidentified host, as well as bacterial, factors contribute to the colonization relationship. These yet-to-be-described interactions may contribute to the predilection of ruminants to serve as a reservoir host for E. coli O157:H7. The results suggest that, in contrast to cattle, sheep, and neonatal pigs, intimin is not required for persistent colonization of E. coli O157:H7 in 12-week-old conventional pigs.

Colonization of Gnotobiotic Piglets by a luxS Mutant Strain of Escherichia coli O157:H7

ABSTRACT Gnotobiotic piglets inoculated with Escherichia coli O157:H7, its luxS mutant derivative, or nonpathogenic E. coli were evaluated for attaching and effacing lesions. Although no differences in clinical symptoms were seen between pigs inoculated with the parent and those inoculated with the luxS mutant, the luxS mutant-inoculated pigs had a lower frequency of attaching and effacing lesions in the spiral colon than parent strain-inoculated pigs.

Development of a real-time polymerase chain reaction assay for detection of Actinobacillus suis in porcine lung

In the current study, the development and validation of a real-time polymerase chain reaction (PCR) assay using a TaqMan-labeled probe for the detection of Actinobacillus suis from porcine lung samples is described. This real-time PCR amplified a 110-bp region of the 23S ribosomal RNA gene from A. suis but not from other bacteria. First, the assay was validated with 183 bacterial strains representing different species of bacteria. Subsequently, 85 porcine lung specimens that were declared A. suis–positive and –negative by bacterial culture and identification were tested to assess whether it can be performed directly on tissue specimens. The bacterial culture results and real-time PCR results agreed across all the samples tested assigning 100% positive and negative predictive values to the PCR. Further, the detection limit of the assay was 380 colony-forming units (CFU) per ml or approximately 1 CFU per reaction. In conclusion, the TaqMan real-time PCR assay described herein is a highly specific, sensitive, and reproducible test, which can be used to detect A. suis DNA in porcine lung specimens, thus providing a timely diagnosis.