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SUMMARYForty-four Paenibacillus larvaesubsp. larvaeisolates cultured from larval smears and honey samples from Australia and honey imported from Argentina were typed using pulsed-field gel electrophoresis (PFGE). Their minimum inhibitory concentrations (MICs) to oxytetracycline (OTC) were also determined using an agar dilution method. PFGE fingerprints of P. l. larvaeDNA generated by the restriction endonuclease Xbal produced 12 distinct PFGE types. Types I, II and VIII comprised the majority of strains (28/44: 63.6%). Type I (9/44: 20.5%) consisted only of isolates cultured from Argentinean honey and Argentinean honey blended with Australian honey, and all these isolates had a MIC to OTC of 0.6 μg/ml. Type II, comprising 22.7% of total (10/44), consisted of isolates from New South Wales (3), Queensland (6) and one isolate originating from a mixture of Argentinean and Australian honey. All these isolates had MICs to OTC of 0.04 or 0.05 μg/ml. Type VIII consisted of 9 (20.5%) isolates, all originating from New South Wales, with MICs to OTC of 0.04 or 0.05 μg/ml. The other PFGE types consisted of 3 or fewer isolates (Type XI, 3 isolates; Types III, V, VI, VII and IX, 2 isolates each; Types IV, X and XII, 1 isolate each). P. l. larvaeisolates of Australian origin and Argentinean origin were all very sensitive to OTC. However, the isolates of Argentinean origin and Argentinean/Australian honey blend origin were less sensitive to OTC (MIC of 0.6 μg/ml) than isolates of Australian origin (MICs of 0.04 or 0.05 μg/ml). Collectively these data indicate that PFGE is an affective means of discriminating between P. l. larvaestrains and could be used to determine whether OTC resistant strains are from local or imported P l. larvaestrains.

ABSTRACTThe 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 (a1, a2, ß, ?, ?, e, ?, ?, ?, ?, and ?) 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-?, and Int-?. The PCR-RFLP assay was used to screen 213 eae-positive E. coliisolates 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-ß, the most commonly identified eaesubtype (82 of 213 [38.5%] isolates), was associated with 21 serotypes, followed by Int-? (39 of 213 [18.3%] isolates; 11 serotypes), Int-? (25 of 213 [11.7%] isolates; 15 serotypes), Int-? (19 of 213 [8.9%] isolates; 9 serotypes), and Int-e (21 of 213 [9.9%] isolates; 5 serotypes). Intimin subtypes a1, a2, ?, ?, ?, µ, ?, and ? were infrequently identified; and Int-? was not detected. Phylogenetic analyses with the Phylip package of programs clustered the intimin subtypes into nine distinct families (a, ß-?, ?, ?, e-?-?, ?-µ, ?, ?, and ?). Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-containing E. colistrains.

ABSTRACTstx2genes 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 ehxAand/or eaeAand 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-EDL933and stx2vhb; 2 isolates contained stx2vhbonly. 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-EDL933and stx2vhb. Three of the four were from separate sporadic cases of HUS, and one was from an unknown source.

Unlike Shiga toxin 2 (stx(2)) genes, most nucleotide sequences of Shiga toxin 1 (stx(1)) 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 stx(1) among STEC isolates derived from human or animal sources. However stx(1OX3), originally identified in an OX3:H8 isolate from a healthy sheep in Germany, differs from other stx(1) subtypes by 43 nucleotides, resulting in changes to 12 amino acid residues, and has been renamed stx(1c). In this study we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that distinguishes stx(1c) from other stx(1) subtypes. The PCR-RFLP assay was used to study 378 stx(1)-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 stx(2)); the combination stx(1), stx(2), 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 stx(1c) 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 stx(1) subtype; 27 (51.9%) of these belonged to serotype O91:H(-). Nineteen of 207 isolates (9.2%) contained both stx(1c) and stx(1) subtypes, and 14 belonged to serotype O75:H8. In marked contrast, 97 of 104 (93.3%) bovine isolates comprising 44 serotypes possessed an stx(1) subtype, 6 isolates possessed stx(1c), and the remaining isolate possessed both stx(1c) and stx(1) subtypes. Ten of 11 (91%) isolates cultured from meat in New Zealand possessed stx(1c) (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 stx(1) recovered from cattle rarely were the same as those isolated from sheep. Although an stx(1c) subtype was never associated with the typical enterohemorrhagic E. coli serogroups O26, O103, O111, O113, and O157, 13 human isolates possessed stx(1c). 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.

ABSTRACTUnlike 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 stx1among 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 stx1subtypes 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 stx1cfrom other stx1subtypes. 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 ehxAwas 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 stx1calone 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 stx1subtype; 27 (51.9%) of these belonged to serotype O91:H-. Nineteen of 207 isolates (9.2%) contained both stx1cand stx1subtypes, and 14 belonged to serotype O75:H8. In marked contrast, 97 of 104 (93.3%) bovine isolates comprising 44 serotypes possessed an stx1subtype, 6 isolates possessed stx1c, and the remaining isolate possessed both stx1cand stx1subtypes. 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 stx1recovered from cattle rarely were the same as those isolated from sheep. Although an stx1csubtype was never associated with the typical enterohemorrhagic E. coliserogroups 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.

ABSTRACTA 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 ehxAwere 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. eaeAwas 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.

ABSTRACTShiga 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 stx2subtype 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 majorstx2subtype 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/OX3asubtype. 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 stx2subtype(s) associates with serotype and may have important epidemiological implications for tracing sources of E. coliduring outbreaks of STEC-associated diseases in humans.

The prevalence of complex Shiga toxin‐producing Escherichia coli(STEC), i.e. STEC containing accessory virulence factors intimin (eaeA) and/or enterohaemorrhagic E. colihaemolysin (ehxA) and their serotypes were determined in diagnostic bovine faecal samples processed during a 3 months period. The presence of complex STEC was determined using PCR and vancomycin‐cefixime‐cefsulodin blood agar (BVCCA) using a dual approach which involved (i) direct culture of faecal samples on BVCCA followed by mutiplex PCR of BVCCA positive colonies and (ii) culture of faecal samples enriched in modified EC (mEC) broth (with a complex STEC profile determined by PCR) on BVCCA followed by multiplex PCR of BVCCA positive colonies. Using both techniques complex STEC were isolated from 23 (18.7%) of the 123 faecal samples. Complex STEC were isolated from 14 faecal samples by direct culture on BVCCA and 13 faecal samples yielded complex STEC by culture of mEC broths with a complex STEC profile on BVCCA. Only four samples were positive using both techniques. The serotypes isolated included O5:H‐, O26:H‐, O26:H11, O91:H21, O111:H‐, O111:H8, O104:H11, O113:H21 and O157:H8. This study confirms that non‐O157 STEC can be isolated from bovine faeces and that they carry types associated with human disease. This work also demonstrates that the use of a dual approach is advisable to increase the likelihood of isolating complex STEC.