Title: The Operon Encoding SubAB a Novel Cytotoxin is Present in US STEC Isolates ACCEPTED

Transcription

1 JCM Accepts, published online ahead of print on February 00 J. Clin. Microbiol. doi:./jcm Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Title: The Operon Encoding SubAB a Novel Cytotoxin is Present in US STEC Isolates Authors: A. Khaitan, BS 1, J. M. Ritchie, Ph.D., A. W. Paton, Ph.D., D. M. Jandhyala, Ph.D. 1, C. M. Thorpe, M.D. 1* ; 1 Tufts-New England Medical Center, Boston, MA, Tufts University School of Medicine, Boston, MA, University of Adelaide, S.A., Australia *corresponding author 0 Washington Street Box 01 Boston, MA 01 Phone: 1--0 Fax: 1-- cthorpe@tufts-nemc.org Keywords: Subtilase cytotoxin, Shiga toxin-producing E. coli, SubAB, non-o1:h Diarrhea-associated hemolytic uremic syndrome (D+HUS) is thought to result from endothelial cell damage following gastrointestinal infection with Shiga toxin-producing Escherichia coli (STEC) []; Shiga toxin (Stx) is regarded as the main virulence factor that causes these microangiopathic sequelae. [,1]. Since STEC species vary in their ability to cause severe disease, other virulence factors may contribute to endothelial damage [1]. It has been hypothesized that the recently discovered AB toxin subtilase cytotoxin (SubAB) may be one of 1

2 these factors [1]. Because of the separate modes of action of SubAB and Shiga toxins, the potential for co-interactions exists [, ] SubAB was first isolated from an Australian non-o1 STEC strain, NK, associated with an outbreak of HUS in children []. In mice, parenteral SubAB causes microangiopathy, suggesting a direct role in endothelial damage [, ]. An estimated % of Australian STEC clinical isolates carry the subab operon; in these strains, its presence is associated with stx and with the gene ehxa []. Furthermore, these strains lack the eae gene encoding the outer membrane adhesion intimin encoded in the locus of enterocyte effacement (LEE) pathogenicity island. The global distribution of SubAB-encoding STEC strains is unknown. Although the epidemiology of D+HUS in Australia differs from the United States, with non-o1 STEC implicated more frequently, we hypothesized that US STEC strains also contain the subab operon. STEC strains originating from US sources were selected from two well-characterized, previously described strain collections [, ]. Previously published multiplex PCR data for stx1, stx, eae, and ehxa, and serogroup/type information were used to select representative strains, allowing assessment of potential associations of the subab operon with these virulence factors and with different O serogroups. Three colonies were assessed in independent PCR runs to ensure correct strain classification, using NK as a positive control and two non-pathogenic E. coli lab strains, HB1 and XLI-Blue, as negative controls. Table 1 shows that US STEC strains of diverse origin harbor suba, confirming that SubAB-expressing organisms are present on the North American subcontinent. Of isolates tested, strains were suba positive; from cattle, from diarrhea patients, and 1 from an environmental water source. Furthermore, these strains have a virulence gene profile similar to suba-positive strains from Australia; of US STEC strains that tested positive for suba had the gene encoding stx toxin variant only, all were eae-negative, and all were ehxa-positive. Only one suba-positive US STEC strain was positive for both stx1 and stx. None of the eaepositive strains had suba. Table summarizes these results. Since these strains were specifically

3 selected to represent different serogroups and virulence markers, no conclusions can be drawn regarding overall US prevalence of SubAB. A potential association of SubAB cytotoxin with human disease has not yet been studied. In the US, non-o1 E. coli account for up to 0%-0% of all STEC infections [], and can cause HUS and HC. Thus, epidemiologic studies should be undertaken to assess a potential role for SubAB in contributing to microangiopathic sequelae following infection with these organisms. It would also be of interest to determine whether SubAB is present in strains from countries where non-01 STEC predominate, such as Argentina, where HUS prevalence is the highest in the world.

4 Table 1. Presence of suba and other putative virulence genes in STEC strains a. Isolate Source Serogroup/type subab stx 1 stx eae ehxa Reference HA HUS patient O b HB HUS patient O b HC HUS patient O b HD HUS patient O b HE HUS patient O b HF HUS patient O b HG HUS patient O b HH HUS patient O b HI HUS patient O b HJ HUS patient O b HK HUS patient O b HL HUS patient O b HM HUS patient O b BA Cattle O b BB Cattle O b BC Cattle O b BD Cattle O b BE Cattle O b BF Cattle O b BG Cattle O b BH Cattle O b BI Cattle O b BJ Cattle O b BK Cattle O b BL Cattle O b BM Cattle O b BN Cattle O b BO Cattle O b BP Cattle O b BQ Cattle OX b BR Cattle OX b BS Cattle OX b BT Cattle OX b BU Cattle NEG b BV Cattle NEG b BW Creek Water NEG b BX Cattle NEG b BY Cattle NEG b 0 Diarrhea patient OX:H c Diarrhea patient OX:H c Diarrhea patient Ow:H c Diarrhea patient O:H c Diarrhea patient O1:HNM c - Diarrhea patient O c - Diarrhea patient O c I-1 Diarrhea patient O1:H c PCR control strains: NK HUS patient O:H d

5 / Ref. strain O e Ref. Strain O1:H e HB1 Lab strain N/A N/A XLIBlue Lab strain N/A N/A a eae, ehxa genotypes and serogrouping/serotyping reported from previous studies. b Ritchie J, et al. 00, ref. ; c Hurley B, et al. 001,ref ; d Paton A et al. 00, ref. ; e Nataro and Kaper, ref.. NEG=non-typeable O antigen, H- = non-typeable H antigen. Table. Summary of virulence factor co-expression of suba-positive and suba-negative STEC. suba-positive Total eae positive 0 eae negative 1 stx only stx 1 and stx 1 1 ehxa positive suba negative

6 References: Bielaszewska M., Karch H. 00. Consequences of enterohemorrhagic E. coli infection for the vascular epithelium. Thromb. Haemost. :1-1.. Hurley B, Thorpe C, Acheson D Shiga toxin translocation across intestinal epithelial cells is enhanced by neutrophil transmigration. Infect. Immun. :1-1.. Johnson K, Thorpe C, Sears C. 00. The emerging clinical importance of non- O1 Shiga toxin-producing Escherichia coli. Clin. Infect. Dis. :1-.. Nataro JP, Kaper JB.. Diarrheagenic E. coli. Clin. Microbiol. Rev. : Paton A, Beddoe T, Thorpe CM, Whisstock J, Wilce M, Rossjohn J, Talbot U, and Paton, J. 00. AB subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP. Nature :-.. Paton A, Paton J. 00. Multiplex PCR for direct detection of Shiga toxigenic Escherichia coli strains producing the novel subtilase cytotoxin. J. Clin. Microbiol. :-.. Paton A, Srimanote P, Talbot U, Wang H, Paton J. 00. A new family of potent AB cytotoxins produced by Shiga toxigenic Escherichia coli. J. Exp. Med. 00: -.. Ritchie J, Wagner P, Acheson D, Waldor M. 00. Comparison of Shiga toxin production by hemolytic-uremic syndrome-associated and bovine-associated Shiga toxin-producing Escherichia coli isolates. App. and Env. Microbiol. : -.. Thorpe C. 00. Shiga toxin-producing Escherichia coli infection. Clin. Infect. Dis. : -.. Montecucco C. and Molinari M. 00. Death of a chaperone. Nature :- 1.