Biofilm – an environment for dissemination of stx genes by 1 transduction 2

MAX 250 WORDS) 14 Dissemination of Shiga toxin-encoding bacteriophages by transduction is the most 15 likely mechanisms for the spread of Stx-encoding genes and the emergence of new Shiga 16 toxin-producing Escherichia coli (STEC). Biofilm have been reported to be a place where 17 horizontal gene transfer by plasmid conjugation and DNA transformation may occur, and in 18 this study horizontal gene transfer by transduction has been demonstrated. Transfer of Stx19 encoding bacteriophages to potentially pathogenic E. coli in biofilm was observed at both 20 20oC and 37oC. The transduction rates were higher at 37oC than at 20oC. To the authors’ 21 knowledge, this study is the first to show lateral gene transfer in biofilm mediated by 22 transduction events with a lysogenic bacteriophage. The study shows that the biofilm 23 environment can be suitable for transduction events and thereby be an environment for the 24 emergence of new pathogenic E. coli. 25 26 27 on O cber 7, 2017 by gest ht://aem . sm . rg/ D ow nladed fom INTRODUCTION 28 Shiga toxin-producing Escherichia coli (STEC) are food borne pathogens that may 29 cause disease ranging from mild diarrhoea to haemorrhagic colitis, and complications such as 30 life-threatening haemolytic-uremic syndrome (HUS) (13). An array of virulence 31 characteristics have been described for STEC (reviewed in (2, 10, 24)), and some of these, 32 such as the eae-encoded adherence factor intimin, are found in other E. coli pathogroups as 33 well. However, the major virulence factors of STEC, and also the characteristic defining the 34 STEC pathogroup, is the production of Shiga toxins (Stx). The stx genes are located within 35 temperate lambdoid bacteriophages that form a heterogeneous family. The host range of Stx36 encoding bacteriophages is highly variable, and bacteriophage transduction into a wide range 37 of E. coli and also other related species in Enterobacteriaceae (e.g. Shigella spp., Citrobacter 38 freundii, Enterobacter cloacae) has been shown in vitro in planktonic cells (5, 7, 16). 39 Moreover, transduction is of importance for the development of emerging pathogenic E. coli, 40 such as for instance the E. coli O104:H4 that caused the large European outbreak in 2011 (15) 41 and possibly also the E. coli O103:H25 that caused an outbreak in Norway in 2006 (17). 42 Stx transduction has been reported to take place in environments such as the 43 gastrointestinal tract of various animals (1, 19, 21) and in the presence of various food 44 matrices at different temperatures (6, 14). Bacterial biofilms are believed to be the natural 45 way of living for the majority of bacterial species. Within bacterial biofilms, vast numbers of 46 bacterial cells live closely together in sessile microbial communities (3). Consequently, the 47 biofilm environment could be an ideal setting for stx transduction. Gene transfer by plasmid 48 conjugation and DNA transformation within biofilm have been reported previously (12). The 49 use of lytic bacteriophages on biofilm as an antibacterial strategy due to their ability to lyse 50 bacteria have also been described (4, 8), but incorporation of bacteriophage carried genes, 51 on O cber 7, 2017 by gest ht://aem . sm . rg/ D ow nladed fom including stx genes, into the bacterial host genome through lysogeny has, to the best of our 52 knowledge, not previously been shown in biofilm. 53 The ability to acquire and incorporate foreign DNA through horizontal gene transfer is 54 an important factor in the evolution of bacteria, including spread of antibiotic resistance genes 55 and virulence genes (9). For the emergence of new STEC serotypes and intergenic spread of 56 stx genes, dissemination of Stx-encoding bacteriophages by transduction is the most likely 57 mechanism. In the present study, we showed that such transduction can indeed occur within 58 biofilms produced by potentially pathogenic E. coli. 59 60 MATERIALS AND METHODS 61 Bacteriophage and bacterial strains 62 One Stx2-encoding bacteriophage Φ731 (Δstx2::cat) (hereafter called Φ731), in which 63 a chloramphenicol resistance gene (chloramphenicol acetyltransferase; cat) has been inserted 64 into stx2, was used for studying transduction events. The original bacteriophage was carried by 65 an E. coli O103:H25 strain from a Norwegian HUS patient (17), but the bacteriophage 66 construct, Φ731, was carried by an E. coli DH5α. In the present study, as further described 67 below, the bacteriophage was transduced into the host E. coli C600 (E. coliC600:Φ731), and 68 this strain was further used as donor strain in the transduction experiments. An eae positive, 69 stx negative ovine E. coli O103:H25 (2006-22-1199-51-2), hereafter called E. coli O103:H25 7