Pathogenicity of enterohemorrhagic Escherichia coli (EHEC) strains depends on various virulence factors, like adhesins (including intimin), ﬂagella, type I ﬁmbriae, curli ﬁbers, hemolysins, and agents required for bioﬁlm formation. However, the pathogenicity is highly enhanced by production of Shiga toxins which are encoded by stx genes located in genomes of Shiga toxin-converting (Stx) prophages. Thus, for maximal EHEC virulence, induction of these prophages is necessary. Although various physical and chemical agents causing Stx prophage induction are known, it is not clear whether any food can stimulate this process. Therefore, the aim of this work was to test whether commonly used drinks might enhance Stx prophage induction in E. coli. It was found that 5% Nestea, but not 5% Coca-Cola or 1% ethanol, caused a signiﬁcant induction of 933W Stx prophage in a bacterial culture in vitro. These results indicate that some commonly used drinks can stimulate Stx prophage induction, potentially enhancing virulence of EHEC. This may have impact on protection of consumers which are infected by these bacteria, and might contribute to choose safe food for such persons. Keywords Enterohemorrhagic Escherichia coli Shiga toxin-converting bacteriophages Commonly used drinks Prophage induction 1 Introduction production, and virulence of STEC, prophage induction (and subsequent expression of phage genes) is necessary Vast majority of Escherichia coli strains are harmless to (Łos ´ et al. 2011). Treatment of STEC infections is prob- humans, and they occur as a part of natural microbiota in lematic, as many antibiotics cause prophage induction, thus the human gut. However, Shiga toxin-producing E. coli enhancing pathogenicity even if bacterial cells are even- (STEC), and particularly a subset of these strains, called tually killed; this is the reason of relatively high mortality enterohemorrhagic E. coli (EHEC) are highly pathogenic among STEC-infected patients and a need for both devel- (Karmali 2017). Usually STEC cause local outbreaks, opment of new treatment options and prevention of Stx which are often severe and dangerous, like that which took prophage induction (Hall et al. 2017). place in Germany in 2011 (Bloch et al. 2012). In the light of the mechanism of STEC pathogenicity, STEC virulence depends on production of Shiga toxins. and the fact that these bacteria are harmless if the Stx These strong toxins are encoded by stx genes, located in phage is kept in lysogenic bacteria in the form of the genomes of Shiga toxin-converting (Stx) prophages. prophage, it appears important to learn about agents which Therefore, for expression of these genes, Shiga toxin might stimulate Stx prophage induction in human intestine. Although there are many physical and chemical factors able to cause this process, including UV irradiation, mit- Electronic supplementary material The online version of this article omycin C, high salt concentrations, EDTA, and hydrogen (https://doi.org/10.1007/s00003-018-1155-z) contains supplementary material, which is available to authorized users. peroxide (Łos ´ et al. 2009; Harris et al. 2012; Imamovic and Muniesa 2012), it is not clear whether particular food & Grzegorz We ˛grzyn products can either stimulate or prevent Stx prophage email@example.com induction. Very recent report on food-related stress Department of Molecular Biology, University of Gdansk, demonstrated that particular environmental factors Wita Stwosza 59, 80-308 Gdansk, Poland 123 126 K. Pierzynowska et al. Fig. 1 Growth of E. coli MG1655 (933W Dstx2::cat-gfp)in LB time 0. OD of bacterial cultures was measured at indicated times. medium at 37 C following supplementation of the medium with Results of 3 independent experiments are shown with error bars different drinks. Following drinks were added to the medium at time indicating SD. Asterisks indicate statistically signiﬁcant differences 0: Coca-Cola (to ﬁnal concentration of 5%), Nestea Lemon (to ﬁnal (p \ 0.05) in the values (calculated as changes in OD during 30 min growth) determined in experiments with mitomycin C relative concentration of 5%) and vodka Zurbo ´ wka (to ﬁnal ethanol concen- to the negative control (water). No statistically signiﬁcant differences tration of 1%). In control experiments, mitomycin C (positive control, were detected between other values added to ﬁnal concentration of 1 lM) and water (negative control, added in the amount of 1:20 of the medium volume) were added at (hydrogen peroxide and acids) could stimulate Stx pro- the tested drinks in our experimental system could roughly phage induction, however, tested stressors, rather than food correspond to their concentrations in human gastrointesti- products, were added to the experimental system (Fang nal tract shortly after consumption. Water and mitomycin et al. 2017). Therefore, we aimed to perform a pilot study C(1 lM), an antibiotic causing prophage induction, were to test whether commonly used drinks may inﬂuence efﬁ- used as negative and positive control, respectively. ciency of Stx prophage induction in E. coli cells. When tested compounds were added to cultures of E. coli lysogenic with 933W Dstx2::cat-gfp, an inhibition of bacterial growth (no increase in absorbance of the cul- 2 Results and discussion ture after 120 min, contrary to earlier measurements) was observed in mitomycin C-treated cells. This may indicate To test effects of various commonly used drinks on efﬁ- prophage induction and subsequent lysis of host cells by ciency of Stx prophage induction in E. coli, MG1655 strain bacteriophages (Fig. 1). Among tested drinks, there were (Jensen 1993) and its derivative carrying a modiﬁed (for only minor (not statistically signiﬁcant) or negligible safety reasons) Stx 933W prophage, 933W Dstx2::cat-gfp effects on bacterial culture growth (Fig. 1). This might be (Nowicki et al. 2015), were employed. Following drinks interpreted as either no induction of prophages or relatively were tested at indicated ﬁnal concentrations in culture low efﬁcient induction. Nevertheless, because even small medium: Coca-Cola (ﬁnal concentration 5%), Nestea (ﬁnal doses of Shiga toxins may be deleterious to humans, more detailed studies were performed. concentration 5%), and vodka Zubro ´ wka (ﬁnal ethanol To assess efﬁciency of prophage induction more concentration 1%). These concentrations were chosen on directly, samples of bacterial cultures were treated with the basis of previously reported volumes of human stomach chloroform, and lysates were titrated (using the MG1655 and intestine (Schiller et al. 2005), and assuming one strain) for the presence of virions. As expected, treatment portion of each drink to be 100 ml. Thus, concentrations of 123 Effects of some commonly used drinks on induction of Shiga toxin-converting prophage in… 127 Fig. 2 Induction of the 933W Dstx2::cat-gfp prophage and its lytic determined at indicated times by phage titration on E. coli MG1655 propagation in E. coli MG1655 (933W Dstx2::cat-gfp)(a), and host strain and calculation of the relative burst size taking into expression of the stx2::cat-gfp fusion gene as assessed by Western account the number of bacterial cells in the culture. At time 0, relative blotting with anti-GFP antibodies (b), after supplementation of the phage burst size was below 0.05 in all tested samples. Results of 3 medium with different drinks. Following drinks were added to the independent experiments are shown with error bars indicating SD. medium at time 0: Coca-Cola (to ﬁnal concentration of 5%), Nestea Asterisk indicates statistically signiﬁcant difference (p \ 0.05) rela- ´ tive to the negative control (water). For Western blotting experiments, Lemon (to ﬁnal concentration of 5%) and vodka Zurbowka (to ﬁnal 1 9 10 cells were harvested by centrifugation, lysed, and the ethanol concentration of 1%). In control experiments, mitomycin C proteins were separated by SDS-PAGE, followed by Western blotting (positive control, added to ﬁnal concentration of 1 lM) and water with anti-GFP antibodies. A representative blot is shown. Positions of (negative control, added in the amount of 1:20 of the medium molecular weight markers are indicated. For loading control, SDS- volume) were added at time 0. Number of liberated phage progeny PAGE gel was stained with Coomassie Brilliant Blue (shown below (which reﬂect frequency of prophage induction and efﬁciency of the blot) phage lytic development) per 1 lysogenic bacterial cell was 123 128 K. Pierzynowska et al. with mitomycin C caused appearance of about 100-times induction and/or phage lytic development. The concentra- more pfu/cell (plaque forming units per cell) in the culture tions of the drinks in the medium (5%), tested in this work, than in negative control experiments (with water; in this may likely occur in the human digestive tract, particularly control, liberated phages represented effects of sponta- shortly after their consumption. Thus, we speculate that neous prophage induction). Addition of Coca-Cola or their effects on prophage induction in STEC infecting vodka did not affect the efﬁciency of prophage induction human intestine might be similar to those observed in the signiﬁcantly (Fig. 2a). However, in the presence of 5% artiﬁcial system employed in this study. However, one Nestea, the number of pfu/cell in the culture was about ten should take into consideration that conditions in human times higher than in the negative control (Fig. 2a). These intestine differ signiﬁcantly from those in our experiments. results indicate that Nestea may stimulate induction of Stx Nevertheless, if our assumption is true, such drinks should prophages in E. coli cells. Expression of stx2::cat-gfp has be avoided if STEC infection is conﬁrmed or even been monitored by Western blotting with anti-GFP anti- suspected. bodies. No signal could be detected at time 0 in any This report provides results of the pilot study suggesting sample. Induction of the prophage by mitomycin C resulted that consumption of particular food products may affect in efﬁcient expression of the fusion gene, as indicated by a Stx prophage induction in STEC strains signiﬁcantly. strong signal on Western blot (Fig. 2b). Such a signal, Therefore, we conclude that further complex studies on though signiﬁcantly less strong, could be observed in effects of a large set of food products (not only drinks but bacteria cultured in the presence of 5% Nestea for 180 min, also other nutrients) on Stx prophage induction should be while signals from cells treated with Coca-Cola, vodka or important to protect consumers infected with STEC strains water were below the detection limit (Fig. 2b). and to assess safety of food to be used by patients suffering The results presented in this report indicate that some from such infections. commonly used drinks, like Nestea, may stimulate induc- Acknowledgements This work was supported by University of tion of Stx prophages in E. coli lysogenic cells. On the Gdan ´ sk (Task Grant no. 530-L140-D242-17-1A). basis of these experiments, it is not possible to distinguish what stage of bacteriophage propagation is more efﬁcient Compliance with ethical standards in the presence of Nestea: frequency of prophage induction or efﬁciency of intracellular lytic development of the phage Conflict of interest The authors declare no conflict of interest. after induction, or both. Nevertheless, irrespective of which Open Access This article is distributed under the terms of the Creative option is true, signiﬁcantly higher number of phage pro- Commons Attribution 4.0 International License (http://creative geny, relative to control experiments, means more efﬁcient commons.org/licenses/by/4.0/), which permits unrestricted use, dis- production of new virions that must be preceded by more tribution, and reproduction in any medium, provided you give efﬁcient expression of phage genes, including stx genes, appropriate credit to the original author(s) and the source, provide a implicating signiﬁcantly more effective production of link to the Creative Commons license, and indicate if changes were made. Shiga toxins. Such increased expression of stx after 933W prophage induction has already been demonstrated exper- imentally (Łos et al. 2009), and is also conﬁrmed here References (Fig. 2b) by monitoring levels of GFP as a product of expression of the fusion gene stx2::cat-gfp. This suggests Bloch SK, Felczykowska A, Nejman-Falenczyk B (2012) Escherichia that consumption of Stx prophage induction-stimulating coli O104:H4 outbreak — have we learnt a lesson from it? Acta drinks by persons infected with STEC might cause either Biochim Pol 59:483–488 Fang Y, Mercer RG, McMullen LM, Ganzle MG (2017) Induction of onset of the disease or more severe symptoms. One might Shiga toxin-encoding prophage by abiotic environmental stress speculate what components of Nestea Lemon, tested in this in food. Appl Environ Microbiol 83:e01378-17 work, could cause stimulation of prophage induction or Hall G, Kurosawa S, Stearns-Kurosawa DJ (2017) Shiga toxin phage lytic development. However, apart from known therapeutics: beyond neutralization. Toxins 9:291 Harris SM, Yue WF, Olsen SA, Hu J, Means WJ, McCormick RJ, Du chemicals included in this drink (sugar, citric acid, sodium M, Zhu MJ (2012) Salt at concentrations relevant to meat citrate, and ascorbic acid), it also contains black tea extract processing enhances Shiga toxin 2 production in Escherichia coli and lemon juice which composition is not strictly deﬁned. O157:H7. Int J Food Microbiol 15:186–192 Recent studies suggested that sodium citrate may increase Imamovic L, Muniesa M (2012) Characterizing RecA independent induction of Shiga toxin2-encoding phages by EDTA treatment. production of Shiga toxins by STEC (Lenzi et al. 2016), PLoS One 7:e32393 however, it was reported previously that citrate inhibits Stx Jensen KF (1993) The Escherichia coli K-12 ‘‘wildtypes’’ W3110 and phage lytic development (Nejman-Falenczyk et al. 2012). MG1655 have an rph frameshift mutation that leads to pyrim- Therefore, further extensive studies are necessary to learn idine starvation due to low pyrE expression levels. J Bacteriol 175:3401–3407 about agents present in Nestea and inﬂuencing prophage 123 Effects of some commonly used drinks on induction of Shiga toxin-converting prophage in… 129 Karmali MA (2017) Emerging public health challenges of Shiga Nejman-Falenczyk B, Golec P, Macia ˛g M, Wegrzyn A, We ˛grzyn G toxin-producing Escherichia coli related to changes in the (2012) Inhibition of development of Shiga toxin-converting pathogen, the population, and the environment. Clin Infect Dis bacteriophages by either treatment with citrate or amino acid 64:371–376 starvation. Foodborne Pathog Dis 9:13–19 Lenzi LJ, Lucchesi PM, Medico L, Burga ´n J, Kru ¨ ger A (2016) Effect Nowicki D, Bloch S, Nejman-Falen ´ czyk B, Szalewska-Pałasz A, of the food additives sodium citrate and disodium phosphate on We ˛grzyn A, We ˛grzyn G (2015) Defects in RNA polyadenylation Shiga toxin-producing Escherichia coli and production of stx- impair both lysogenization by and lytic development of Shiga phages and Shiga toxin. Front Microbiol 7:992 toxin-converting bacteriophages. J Gen Virol 96:1957–1968 Łos ´ JM, Łos ´ M, We ˛grzyn A, We ˛grzyn G (2009) Differential Schiller C, Fro ¨ hlich CP, Giessmann T, Siegmund W, Mo ¨ nnikes H, efﬁciency of induction of various lambdoid prophages respon- Hosten N, Weitschies W (2005) Intestinal ﬂuid volumes and sible for production of Shiga toxins in response to different transit of dosage forms as assessed by magnetic resonance induction agents. Microb Pathogen 47:289–298 imaging. Aliment Pharmacol Ther 22:971–979 Łos ´ JM, Łos ´ M, We ˛grzyn G (2011) Bacteriophages carrying Shiga toxin genes: genomic variations, detection and potential treat- ment of pathogenic bacteria. Future Microbiol 6:909–924
Journal für Verbraucherschutz und Lebensmittelsicherheit – Springer Journals
Published: Feb 2, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera