Michael A. Hornitzky

Distribution of Intimin Subtypes among Escherichia coli Isolates from Ruminant and Human Sources

ABSTRACT The intimin gene eae, located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenic Escherichia coli (EPEC) and some Shiga toxin-producing E. coli (STEC) strains from all other pathotypes of diarrheagenic E. coli. EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolytic-uremic syndrome and hemorrhagic colitis. Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (α1, α2, β, γ, κ, ε, η, ι, λ, θ, and ζ) and three new intimin genes that show less than 95% nucleotide sequence identity with existing intimin types. We designated these new intimin genes Int-μ, Int-ν, and Int-ξ. The PCR-RFLP assay was used to screen 213 eae-positive E. coli isolates derived from ovine, bovine, and human sources comprising 60 serotypes. Of these, 82 were STEC isolates, 89 were stx-negative (stx−) and ehxA-positive (ehxA+) isolates, and 42 were stx− and ehxA-negative isolates. Int-β, the most commonly identified eae subtype (82 of 213 [38.5%] isolates), was associated with 21 serotypes, followed by Int-ζ (39 of 213 [18.3%] isolates; 11 serotypes), Int-θ (25 of 213 [11.7%] isolates; 15 serotypes), Int-γ (19 of 213 [8.9%] isolates; 9 serotypes), and Int-ε (21 of 213 [9.9%] isolates; 5 serotypes). Intimin subtypes α1, α2, κ, λ, ξ, μ, ν, and ι were infrequently identified; and Int-η was not detected. Phylogenetic analyses with the Phylip package of programs clustered the intimin subtypes into nine distinct families (α, β-ξ, γ, κ, ε-η-ν, ι-μ, λ, θ, and ζ). Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-containing E. coli strains.

stx1c Is the Most Common Shiga Toxin 1 Subtype among Shiga Toxin-Producing Escherichia coli Isolates from Sheep but Not among Isolates from Cattle

ABSTRACT Unlike Shiga toxin 2 (stx2) genes, most nucleotide sequences of Shiga toxin 1 (stx1) genes from Shiga toxin-producing Escherichia coli (STEC), Shigella dysenteriae, and several bacteriophages (H19B, 933J, and H30) are highly conserved. Consequently, there has been little incentive to investigate variants of stx1 among STEC isolates derived from human or animal sources. However stx1OX3, originally identified in an OX3:H8 isolate from a healthy sheep in Germany, differs from other stx1 subtypes by 43 nucleotides, resulting in changes to 12 amino acid residues, and has been renamed stx1c. In this study we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that distinguishes stx1c from other stx1 subtypes. The PCR-RFLP assay was used to study 378 stx1-containing STEC isolates. Of these, 207 were isolated from sheep, 104 from cattle, 45 from humans, 11 from meat, 5 from swine, 5 from unknown sources, and 1 from a cattle water trough. Three hundred fifty-five of the 378 isolates (93.9%) also possessed at least one other associated virulence gene (ehxA, eaeA, and/or stx2); the combination stx1, stx2, and ehxA was the most common (175 of 355 [49.3%]), and 90 of 355 (25.4%) isolates possessed eaeA. One hundred thirty-six of 207 (65.7%) ovine isolates possessed stx1c alone and belonged to 41 serotypes. Seventy-one of 136 (52.2%) comprised the common ovine serotypes O5:H−, O128:H2, and O123:H−. Fifty-two of 207 isolates (25.1%) possessed an stx1 subtype; 27 (51.9%) of these belonged to serotype O91:H−. Nineteen of 207 isolates (9.2%) contained both stx1c and stx1 subtypes, and 14 belonged to serotype O75:H8. In marked contrast, 97 of 104 (93.3%) bovine isolates comprising 44 serotypes possessed an stx1 subtype, 6 isolates possessed stx1c, and the remaining isolate possessed both stx1c and stx1 subtypes. Ten of 11 (91%) isolates cultured from meat in New Zealand possessed stx1c (serotypes O5:H−, O75:H8/H40, O81:H26, O88:H25, O104:H−/H7, O123:H−/H10, and O128:H2); most of these serotypes are commonly recovered from the feces of healthy sheep. Serotypes containing stx1 recovered from cattle rarely were the same as those isolated from sheep. Although an stx1c subtype was never associated with the typical enterohemorrhagic E. coli serogroups O26, O103, O111, O113, and O157, 13 human isolates possessed stx1c. Of these, six isolates with serotype O128:H2 (from patients with diarrhea), four O5:H− isolates (from patients with hemolytic-uremic syndrome), and three isolates with serotypes O123:H− (diarrhea), OX3:H8 (hemolytic-uremic syndrome), and O81:H6 (unknown health status) represent serotypes that are commonly isolated from sheep.

Virulence Properties and Serotypes of Shiga Toxin-Producing Escherichia coli from Healthy Australian Slaughter-Age Sheep

ABSTRACT A group of 1,623 ovine fecal samples recovered from 65 geographically distinct mutton sheep and prime lamb properties across New South Wales, Australia, were screened for the presence of Shiga toxin-producing Escherichia coli (STEC) virulence factors (stx1, stx2,eaeA, and ehxA). A subset was cultured for STEC isolates containing associated virulence factors (eaeAand/or ehxA), which were isolated from 17 of 20 (85%) and 19 of 20 (95%) tested prime lamb and mutton sheep properties, respectively. STEC isolates containing stx1,stx2, and ehxA were most commonly isolated (19 of 40 flocks; 47.5%), and this profile was observed for 10 different serotypes. Among 90 STEC isolates studied, the most common serotypes were O91:H− (22 isolates [24.4%]), O5:H− (16 isolates [17.8%]), O128:H2 (11 isolates [12.2%]), O123:H− (8 isolates [8.9%]), and O85:H49 (5 isolates [5.6%]). Two isolates (2.2%) were typed as O157:H−. A total of 78 of 90 STEC isolates (86.7%) expressed Shiga toxin in Vero cell culture and 75 of 84ehxA-positive isolates (89.3%) expressed enterohemolysin on washed sheep blood agar. eaeA was observed in 11 of 90 (12.2%) ovine STEC isolates, including serotypes O5:H−, O84:H−, O85:H49, O123:H− O136:H40, and O157:H−. Although only 2 of 90 isolates were typed as O157:H−, the predominant serotypes recovered during this study have been recovered from human patients with clinical disease, albeit rarely.

The common ovine Shiga toxin 2-containing Escherichia coli serotypes and human isolates of the same serotypes possess a Stx2d toxin type

ABSTRACT Shiga toxin 2 (Stx2) has been reported as the main Shiga toxin associated with human disease. In addition, the Stx2 toxin type can have a profound impact on the degree of tissue damage in animal models. We have characterized the stx2 subtype of 168 Shiga toxin-producing Escherichia coli (STEC) isolates of which 146 were derived from ovine sources (principally feces and meat) and 22 were isolated from humans. The ovine STEC isolates were of serotypes that have been shown to occur commonly in the gastrointestinal tract of healthy sheep. The majorstx2 subtype in the ovine isolates was shown to be stx2d-Ount (119 of 146 [81.5%]) and was predominantly associated with serotypes O75:H−/H8/H40, O91:H−, O123:H−, O128:H2, and OR:H2. However, 17 of 18 (94.4%) ovine isolates of serotype O5:H−possessed a stx2d-O111/OX3a subtype. Furthermore, STEC isolates of serotypes commonly found in sheep and recovered from both clinical and nonclinical human infections also contained a stx2d(stx2d-Ount/O111/OX3a) subtype. These studies suggest that a specific stx2 subtype(s) associates with serotype and may have important epidemiological implications for tracing sources of E. coli during outbreaks of STEC-associated diseases in humans.

Bovine Non-O157 Shiga Toxin 2-Containing Escherichia coli Isolates Commonly Possess stx2-EDL933 and/or stx2vhb Subtypes

ABSTRACT stx 2 genes from 138 Shiga toxin-producing Escherichia coli (STEC) isolates, of which 127 were of bovine origin (58 serotypes) and 11 of human origin (one serotype; O113:H21), were subtyped. The bovine STEC isolates from Australian cattle carried ehxA and/or eaeA and predominantly possessed stx2-EDL933 (103 of 127; 81.1%) either in combination with stx2vhb (32 of 127; 25.2%) or on its own (52 of 127; 40.4%). Of 22 (90.9%) bovine isolates of serotype O113:H21, a serotype increasingly recovered from patients with hemolytic uremic syndrome (HUS) or hemorrhagic colitis, 20 contained both stx2-EDL933 and stx2vhb; 2 isolates contained stx2vhb only. Although 7 of 11 (63.6%) human O113:H21 isolates associated with diarrhea possessed stx2-EDL933, the remaining 4 isolates possessed a combination of stx2-EDL933 and stx2vhb. Three of the four were from separate sporadic cases of HUS, and one was from an unknown source.

Virulence Properties and Serotypes of Shiga Toxin-Producing Escherichia coli from Healthy Australian Cattle

ABSTRACT The virulence properties and serotypes of complex Shiga toxin-producing Escherichia coli (cSTEC) were determined in two studies of healthy cattle in eastern Australia. In the first, a snapshot study, 84 cSTEC isolates were recovered from 37 of 1,692 (2.2%) fecal samples collected from slaughter-age cattle from 72 commercial properties. The second, a longitudinal study of three feedlots and five pasture beef properties, resulted in the recovery of 118 cSTEC isolates from 104 animals. Of the 70 serotypes identified, 38 had not previously been reported.

Serotypes and virulence gene profiles of shiga toxin-producing Escherichia coli strains isolated from feces of pasture-fed and lot-fed sheep.

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains possessing genes for enterohemolysin (ehxA) and/or intimin (eae), referred to here as complex STEC (cSTEC), are more commonly recovered from the feces of humans with hemolytic uremic syndrome and hemorrhagic colitis than STEC strains that do not possess these accessory virulence genes. Ruminants, particularly cattle and sheep, are recognized reservoirs of STEC populations that may contaminate foods destined for human consumption. We isolated cSTEC strains from the feces of longitudinally sampled pasture-fed sheep, lot-fed sheep maintained on diets comprising various combinations of silage and grain, and sheep simultaneously grazing pastures with cattle to explore the diversity of cSTEC serotypes capable of colonizing healthy sheep. A total of 67 cSTEC serotypes were isolated, of which 21 (31.3%), mainly isolated from lambs, have not been reported. Of the total isolations, 58 (86.6%) were different from cSTEC serotypes isolated from a recent study of longitudinally sampled healthy Australian cattle (M. Hornitzky, B. A. Vanselow, K. Walker, K. A. Bettelheim, B. Corney, P. Gill, G. Bailey, and S. P. Djordjevic, Appl. Environ. Microbiol. 68:6439-6445, 2002). Our data suggest that cSTEC serotypes O5:H−, O75:H8, O91:H−, O123:H−, and O128:H2 are well adapted to colonizing the ovine gastrointestinal tract, since they were the most prevalent serotypes isolated from both pasture-fed and lot-fed sheep. Collectively, our data show that Australian sheep are colonized by diverse cSTEC serotypes that are rarely isolated from healthy Australian cattle.

Distribution of Class 1 Integrons with IS26-Mediated Deletions in Their 3′-Conserved Segments in Escherichia coli of Human and Animal Origin

Class 1 integrons play a role in the emergence of multi-resistant bacteria by facilitating the recruitment of gene cassettes encoding antibiotic resistance genes. 512 E. coli strains sourced from humans (n = 202), animals (n = 304) and the environment (n = 6) were screened for the presence of the intI1 gene. In 31/79 integron positive E. coli strains, the gene cassette regions could not be PCR amplified using standard primers. DNA sequence analysis of 6 serologically diverse strains revealed atypical integrons harboured the dfrA5 cassette gene and only 24 bp of the integron 3′-conserved segment (CS) remained, due to the insertion of IS26. PCR targeting intI1 and IS26 followed by restriction fragment length polymorphism (RFLP) analysis identified the integron-dfrA5-IS26 element in 27 E. coli strains of bovine origin and 4 strains of human origin. Southern hybridization and transformation studies revealed the integron-dfrA5-IS26 gene arrangement was either chromosomally located or plasmid borne. Plasmid location in 4/9 E. coli strains and PCR linkage of Tn21 transposition genes with the intI1 gene in 20/31 strains, suggests this element is readily disseminated by horizontal transfer.

Development of a hemi-nested PCR assay for the specific detection of Melissococcus pluton

SUMMARYA pair of oligonucleotide primers (MP1 and MP2) were used for the polymerase chain reaction (PCR) amplification of a 486 base pair (bp) fragment of the 16S rRNA gene of 26 geographically diverse Australian Melissococcus pluton (causative agent of European foulbrood) isolates. PCR primers spanning a region of the 16S rRNA gene from position 893–1377 failed to amplify a product when template DNA from a wide range of pathogenic and saprophytic bacteria were used including Paenibacillus larvae, Paenibacillus alvei, Enterococcus faecium and Spiroplasma melliferum. The PCR did, however, reliably amplify a 486 bp fragment (when the annealing temperature was lowered by 5°C) using template DNA isolated from the phylogenetically-related bacterium Enterococcus faecalis. PCR amplicons generated from E. faecalis and M. pluton were readily distinguished by digestion with the restriction endonuclease Hinfl and electrophoresis in 1.5% agarose or by electrophoresis in 1% agarose containing bis-benzidene/polyethylen...

DNA restriction endonuclease profiles and typing of geographically diverse isolates of Bacillus larvae

SUMMARYDNA isolated from each of 20 Bacillus larvae isolates of geographically diverse origin (17 from Australia, one each from Fiji, Mexico and New Zealand) and cultured on brain heart infusion agar was digested with a range of restriction endonucleases. Based upon the degree of similarity in restriction endonuclease fragment patterns (REFP) produced by the restriction endonuclease Cfo I, five clonal types of B. larvae were identified. REFP were resolved on 3.5% Polyacrylamide gels and visualized by silver staining. All the isolates showed many similarities in their REFP, however, those isolates cultured from geographically localized regions showed the greatest similarity. The superior resolving capabilities of polyacrylamide enabled these isolates to be further sub-typed according to minor variations in REFP.