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60Co irradiation of Shiga toxin (Stx)-producing Escherichia coli induces Stx phage
FEMS Microbiology Letters 222 (2003) 115^121
www.fems-microbiology.org
60
Co irradiation of Shiga toxin (Stx)-producing Escherichia coli induces Stx phage
Tatsuo Yamamoto , Seiichi Kojio, Ikue Taneike, Saori Nakagawa, Nobuhiro Iwakura, Noriko Wakisaka-Saito Division of Bacteriology, Department of Infectious Disease Control and International Medicine, Niigata University Graduate School of Medical and Dental Sciences, 757 Ichibanchou, Asahimachidori, Niigata, Japan Received 22 January 2003 ; received in revised form 10 March 2003; accepted 25 March 2003 First published online 16 April 2003
Abstract Shiga toxin (Stx)-producing Escherichia coli (STEC), an important cause of hemolytic uremic syndrome, was completely killed by 60 Co irradiation at 1Ul03 gray (1 kGy) or higher. However, a low dose of irradiation (0.1^0.3 kGy) markedly induced Stx phage from STEC. Stx production was observed in parallel to the phage induction. Inactivation of Stx phage required a higher irradiation dose than that for bacterial killing. Regarding Stx, cytotoxicity was susceptible to irradiation, but cytokine induction activity was more resistant than Stx phage. The findings suggest that (1) although 60 Co irradiation is an effective means to kill the bacteria, it does induce Stx phage at a lower irradiation dose, with a risk of Stx phage transfer and emergence of new Stx-producing strains, and (2) irradiation differentially inactivates some activities of Stx. 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords :
60
Co irradiation ; Shiga toxin-producing Escherichia coli ; Shiga toxin phage; Shiga toxin; Induction; Inactivation
1. Introduction Shiga toxin (Stx)-producing Escherichia coli (STEC) continues to be an important food-borne bacterial pathogen threatening public health [1]. In Japan in 1996, large outbreaks of STEC infection occurred with 17 877 people infected and 12 fatalities [2,3]. STEC is generally associated with intestinal symptoms and also with serious systemic complications such as hemolytic uremic syndrome, especially in the young and elderly [4,5]. STEC includes not only serotype O157:H7 (the most frequently isolated serotype) but also some other serotypes (e.g.) O26 and O86 [1,5,6]. The most common source of STEC O157:H7 infection is undercooked ground beef [5,7]. STEC O157:H7 is isolated from cattle, which are considered as a reservoir of STEC O157:H7 [8,9]. STEC elaborates Stx1 and/or Stx2, both of which are encoded by prophages lysogenized in STEC strains [1,10].
Cobalt-60 (60 Co) irradiation has been employed for preservation of some foods [11,12]. In practice, ‘low’ dose, up to 1Ul03 gray (1 kGy), is used to delay physiological processes (such as sprouting of fresh fruits), and ‘high’ dose, greater than 10 kGy, is used for avoidance of o¡-odors of meat, seafood and others (although such doses do not kill bacterial spores) [11]. In Japan, irradiation at 0.06^0.145 kGy ( 6 0.15 kGy) is used to inhibit sprouting of potatoes [13]. It has not been investigated whether 60 Co irradiation results in Stx phage induction. In this study, we investigated the killing of STEC strains belonging to various serotypes, induction of Stx phage and inactivation of puri¢ed Stx phage and Stx activity by 60 Co irradiation.
2. Materials and methods 2.1. Bacterial strains
* Corresponding author. Tel. : +81 (25) 227 2050; Fax : +81 (25) 227 0762. E-mail address : [email protected] (T. Yamamoto).
Sixteen STEC human strains were tested. They included ¢ve O157:H7 strains (two Stx1þ Stx2þ strains and three Stx2þ strains), ¢ve O26:H11 strains (Stx1þ ), two
0378-1097 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00259-3
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O111:HUT (H, untypable) strains (an Stx1þ Stx2þ strain and an Stx1þ strain), one O128:H2 strain (Stx1þ Stx2þ ), one O128:H12 strain (Stx1þ Stx2þ ), one O145:H3 strain (Stx1þ Stx2þ ) and one O86:H3 strain (Stx2þ ) [6]. The strains were isolated from outbreaks in Japan in 1991 or 1996 and from sporadic cases in Japan in 1996 or 1999. Bovine O157:H7 strains used were two Stx1þ Stx2þ strains and three Stx2þ strains; they were isolated from feces. 2.2. Media and bacterial growth Nutrient agar (Eiken, Tokyo, Japan) was used as solid media. Luria^Bertani (LB) broth (Difco Laboratories, Detroit, MI, USA) was used as liquid media, which was inoculated and incubated at 37‡C for 12^18 h with agitation. TC-SMAC (Denka Seiken Co., Tokyo, Japan) was used as a selective agar plate for STEC O157:H7 strains. 2.3.
60
Co irradiation of STEC strains
Bacteria grown in LB broth at 37‡C were suspended in phosphate-bu¡ered saline (PBS, pH 7.4) at a concentration of ca. 1Ul09 colony-forming units (CFU) ml31 . The bacterial suspension in a plastic tube (diameter 9 mm) was irradiated by 60 Co at 1Ul02 to 3Ul03 Gy (1Ul04 to 3Ul05 rad) at room temperature (ca. 15‡C), and viable counts (CFU) were determined on agar plates. In some experiments, small pieces (1 g) of 60 Co (or UV)pre-irradiated beef were immersed in suspensions of STEC (200 Wl of 1Ul09 CFU ml31 ), and subjected to 60 Co irradiation. The small beef pieces were then placed in PBS (20 ml), homogenized, and viable cell counts in the homogenates were determined on TC-SMAC agar plates. 2.4. Plasmid analysis Plasmid DNA in STEC O157:H7 cells (from a 2-ml portion of the irradiated or non-irradiated samples) was analyzed essentially by a previously described method [14] with some modi¢cations [15]. Plasmid DNA was electrophoresed in 0.5% agarose, stained with ethidium bromide and analyzed with an image analyzer. 2.5. Phage induction, phage plaque assay and Stx assay Stx2þ O157:H7 strain (OB1) and Stx2þ O86:H3 strain (1076) were grown to the exponential phase (ca. 5Ul08 CFU ml31 ) in LB broth at 37‡C, and the bacterial cells were suspended in equal volumes of PBS, followed by 60 Co irradiation. The bacterial cells were then centrifuged and resuspended in the same volume of fresh LB broth, followed by incubation for 30^180 min at 37‡C. The resultant bacterial culture was divided into two parts. One part was centrifuged and the supernatant was ¢l-
trated through a membrane with a pore size of 0.45 Wm, and the ¢ltrate was subjected to phage titration using E. coli indicator strain C600. CaCl2 was added at 10 mM. The numbers of phage plaques that developed in LB soft agar (0.5%) plates (plaque-forming units, PFU) were counted. The plaques (at least 20 plaques on each plate) were examined for the Stx2 gene by PCR assay [16] ; the plaques developed were all from Stx2 phage. The remaining part was subjected to soni¢cation and centrifugation. The supernatant was ¢ltrated through a membrane with a pore size of 0.45 Wm, two-fold serial dilutions of the supernatant were made. Stx was determined by the reverse passive latex agglutination test using a VT detection kit (Denka Seiken). The Stx titers represented the highest dilution to yield positive results. 2.6. Preparation and
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Co irradiation of Stx2 phage
Stx2 phages from STEC strains OB1 and l076 were adsorbed onto E. coli C600 cells in LB broth containing 10 mM CaCl2 at a multiplicity of infection of 0.01^0.1 for 20 min at 37‡C, followed by incubation for 8 h at 37‡C in LB soft agar (0.5%) containing 10 mM CaCl2 . Stx2 phages were collected, and puri¢ed through CsCl density gradient centrifugation as described [10]. Puri¢ed Stx phage was suspended in LB broth containing 10 mM CaCl2 and irradiated by 60 Co as above. 2.7. Preparation and
60
Co irradiation of Stx2
Stx2 produced from E. coli strain Tp8 was puri¢ed to homogenity as described previously [17,18]. The Stx2 preparation (0.2 mg protein per ml in PBS) contained no detectable endotoxin (less than 0.05 endotoxin units per ml) contamination as determined by a limulus amebocyte lysate assay. Puri¢ed Stx in PBS (500 ng ml31 ) was irradiated by 60 Co as above. 2.8. Cytotoxicity assay The cytotoxicity of Stx to Vero cells was examined as described previously [19,20]. The cytotoxic titers of the irradiated or non-irradiated puri¢ed Stx2 samples were determined using two-fold serial dilutions. The highest toxin dilution that caused lysis of 50% of the Vero cell monolayer was taken as the titer of each sample. 2.9. Preparation of human peripheral blood monocytes Human mononuclear cells were prepared from peripheral blood of healthy volunteers using Ficoll-Paque (Gibco BRL, Grand Island, NY, USA) gradient centrifugation. Adherent monocytes were then obtained after incubation in 24-well culture plates (A/S Nunc, Roskilde, Denmark) for 60 min at 37‡C in a CO2 incubator with subsequent washing under high pressure [21].
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60
Co irradiation
Origin and serotype of STEC (number)
Humans O157 :H7 (n = 5) O26:H11 (n = 5) O111 :HUT (n = 2) O128 (n = 2) O145 :H3 (n = 1) O86:H3 (n = 1) Bovine O157 :H7 (n = 5)
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Viable cells (CFU ml31 ) after
60
3U102
1U103
3U103
(6.6 S 5.9)U107 (2.3 S 0.4)U108 (3.6 S 2.0)U107 (4.4 S 4.5)U107 1.8U107 2.8U108
(9.0 S 14.8)U105 (8.5 S 4.1)U106 (1.8 S 2.4)U104 (1.3 S 1.3)U104 6U103 1.5U107
6 1.0 6 1.0 6 1.0 6 1.0 6 1.0 6 1.0
6 1.0 6 1.0 6 1.0 6 1.0 6 1.0 6 1.0
(1.3 S 0.1)U108
(2.6 S 3.5)U106
6 1.0
6 1.0
0
1U10
(9.7 S 1.1)U108 (1.1 S 0.2)U109 (9.1 S 1.1)U108 (1.2 S 0.2)U109 9.9U108 1.2U109 (1.2 S 0.4)U109
2.10. Cytokine assay
Co irradiation at: (Gy)
2
0.3 kGy and 1 kGy, respectively, and was barely detected after 3-kGy irradiation ( 6 4.5%).
The human monocytes (5Ul05 cells ml31 ) prepared as above were stimulated with irradiated or non-irradiated Stx2 (5 ng ml31 ) for 18 h at 37‡C in a CO2 incubator. After incubation, tumor necrosis factor K (TNF-K) in the supernatants was assayed with enzyme-linked immunosorbent assay kits (Genzyme, Cambridge, MA, USA) in accordance with the manufacturer’s instructions.
3.3. Stx phage induction by
60
Co irradiation
An outbreak-derived Stx2þ O157:H7 strain (OB1) and a sporadic case-derived Stx2þ O86:H3 strain (1076) were irradiated by various doses of 60 Co, and the irradiated
3. Results 3.1. Killing of STEC human and bovine strains by 60 Co irradiation STEC human strains including outbreak-derived O157:H7 strains and STEC bovine strains at a concentration of ca. 1Ul09 CFU ml31 in PBS were irradiated by 60 Co at 0.1, 0.3, 1 and 3 kGy, and then viable cell counts in each sample were determined (Table 1). All the STEC strains were killed (the viable counts decreased by 1U109 or more) by 60 Co irradiation at 1 kGy or higher. After irradiation at 0.1 kGy and 0.3 kGy, the viable cell counts decreased by about 1U101 to 1U102 and by about 1U102 to 1U105 , respectively. In beef, a higher irradiation dose was required, and the irradiation dose required to completely kill the STEC O157:H7 strains (isolated from an outbreak as well as from bovine) was 3 kGy (Fig. 1). At 1 kGy, 103 ^104 fold more cells were still alive, compared with those in PBS. 3.2. Destruction of DNA present in STEC by 60 Co irradiation The STEC human and bovine strains had a large plasmid(s) of ca. 90 kbp in size. Those large plasmids present in bacterial cells as a covalently closed circular form were destroyed by 60 Co irradiation ; the covalently closed circular form decreased to 70.5%, 37.7% and 14.6% at 0.1 kGy,
Fig. 1. Inactivation of human and bovine O157:H7 strains of STEC, suspended in PBS or contaminating beef, by 60 Co irradiation. Open symbols represent residual bacterial viable counts (CFU ml31 ) after irradiation at 0.1, 0.3, 1 or 3 kGy for STEC suspended in PBS. Initial viable counts of untreated samples were ca. 1Ul09 CFU ml31 . Closed symbols represent residual bacterial viable counts (CFU ml31 ) after irradiation at 0.1, 0.3, 1 or 3 kGy for STEC contaminating beef. In this case, small pieces of beef (1 g) were mixed with ca. 2Ul08 CFU of STEC, and after irradiation, the small beef pieces were placed in 20 ml of PBS, homogenized, and viable cell counts in the homogenates were determined. Open circle with unbroken line represents data from outbreak-derived human strain OB1 suspended in PBS. Open triangle with broken line represents data from bovine strain V29 suspended in PBS. Closed circle with unbroken line represents data from strain OB1 contaminating beef. Closed triangle with broken line represents data from strain V29 contaminating beef. An asterisk shows that the data are below the detection limit ( 6 1 CFU ml31 ).
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bacterial cells were incubated in a fresh medium for 2 h at 37‡C. The resultant culture supernatants were then examined for Stx phage and Stx. 60 Co irradiation of both strains OB1 and 1076 increased Stx2 phage induction (Fig. 2). The phage induction was most marked at 0.1^0.3 kGy (Fig. 2), with STEC viable counts being decreased to 12.5% to 0.1% (for strain OB1) or 23.3% to 1.3% (for strain 1076). The phage induction reached a level 4.1U102 -fold higher (at 0.1 kGy for strain OB1) or 8.3U103 -fold higher (at 0.3 kGy for strain 1076) than the frequency of spontaneous induction. Stx production was observed in parallel to the phage induction (Fig. 2). Similar Stx production was observed,
Fig. 3. Time course of Stx phage propagation and Stx production after 60 Co irradiation in human O157:H7 and O86:H3 strains of STEC. Bacterial strains used were an outbreak-derived, Stx2þ O157:H7 strain (OB1) in A and a sporadic case-derived, Stx2þ O86:H3 strain (1076) in B. Bacterial cells were suspended in PBS at a concentration of ca. 5Ul08 CFU ml31 , and irradiated at 0.3 kGy for phage induction (at this irradiation, viable counts decreased to 0.1% for strain OB1 and 1.3% for strain 1076). The irradiated cells were then incubated in fresh LB broth at 37‡C for phage propagation and Stx production. Stx2 phage (open circle) and Stx2 (open square) in the culture were periodically examined at the indicated time. An asterisk shows that the data are below the detection limit ( 6 1 PFU ml31 or no detectable Stx2 in the original, undiluted samples). Fig. 2. Stx phage induction and Stx production in 60 Co-irradiated human O157:H7 and O86:H3 strains of STEC. Bacterial strains used were an outbreak-derived, Stx2þ O157:H7 strain (OB1) in A and a sporadic case-derived, Stx2þ O86:H3 strain (1076) in B. Bacterial cells suspended in PBS (ca. 5Ul08 CFU ml31 ) were irradiated at 0.01, 0.03, 0.1, 0.3, 1, 3, 10 or 20 kGy, and each sample was divided into two parts. One part was used for viable cell counting (open triangle). The irradiated cells in the remaining part were incubated in fresh LB broth for 2 h at 37‡C and then Stx2 phage (closed circle) and Stx2 (closed square) in the culture were examined. An asterisk shows that the data are below the detection limit ( 6 1 CFU ml31 , 6 1 PFU ml31 or no detectable Stx2 in the original, undiluted samples).
even when the bacterial cultures were sonicated and the supernatant was examined. The STEC cells, irradiated by 60 Co at 0.3 kGy, were incubated in a fresh medium for various times at 37‡C, and then Stx phage titers and Stx titers were examined (Fig. 3). The Stx phage propagation as well as Stx production nearly plateaued after 120 min (for strain OB1) or 90 min (for strain 1076).
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At a higher irradiation dose, the frequency of phage induction (as well as Stx production) decreased drastically, and no detectable phage induction (or Stx production) was observed at 10 kGy or higher (Fig. 2). 3.4. Inactivation of Stx phage by
60
Co irradiation
Next, Stx2 phages, originating from strains OB1 and 1076, were puri¢ed and irradiated by various doses of 60 Co (Fig. 4). The phages were more resistant to 60 Co irradiation than STEC, and complete inactivation of the phage activity required 10 kGy or higher (Fig. 4). 3.5. Inactivation of the Stx activity by
60
Co irradiation
Puri¢ed Stx2 was irradiated by various doses of 60 Co (Fig. 5). The toxin activity was measured using two methods, measurement of cytotoxicity to Vero cells and measurement of in£ammatory cytokine production from human peripheral blood monocytes. When cytotoxic activity against Vero cells was used as the index, the activity decreased drastically by 212 or more at 1-kGy irradiation. In contrast, the activity for in£ammatory cytokine production in human peripheral blood monocytes was resistant to irradiation, and no signi¢cant decrease was observed at 1 kGy. Even at 20-kGy irradiation, decrease was only about 12%.
Fig. 4. Inactivation of puri¢ed Stx2 phage by 60 Co irradiation. Stx2 phages used originated from an outbreak-derived, Stx2þ O157 :H7 strain OB1 (open circle) and a sporadic case-derived, Stx2þ O86:H3 strain 1076 (closed circle). Phage samples were irradiated at 0.1, 0.3, 1, 3, 10 or 20 kGy, and then PFU were determined by the soft agar method. An asterisk shows that the data are below the detection limit ( 6 1 PFU ml31 ).
Fig. 5. Di¡erential inactivation of biological activities of puri¢ed Stx2 by 60 Co irradiation. Stx2 samples in PBS (500 ng ml31 ) were irradiated at 0.01, 0.03, 0.1, 0.3, 1, 3, 10 or 20 kGy, and then the cytotoxic activity against Vero cells (closed circle) and the activity to induce TNF-K from human peripheral blood monocytes (open circle) were examined. An asterisk shows that the data are below the detection limit (no detectable cytotoxicity in the original, undiluted samples).
4. Discussion In this study, we demonstrated that 60 Co irradiation was useful for killing the outbreak-derived as well as sporadic case-derived STEC strains (regardless of their serotypes). However, we found that 60 Co irradiation markedly induces Stx phages under certain conditions. Phage induction was most marked at 0.1^0.3 kGy, with STEC viable counts being decreased to about 1% (and the STEC 90-kbp plasmid DNA being destroyed by about 50%). Under the condition (at 0.1^0.3 kGy), the Stx phage induction reached levels ca. 103 ^104 -fold higher than the frequency of spontaneous induction. Stx production was observed in parallel to the phage induction. The observation is consistent with the previous notion that Stx prophages are induced with agents that damage DNA or inhibit DNA replication [10,22^24]. Zhang et al. [23] have demonstrated that administration of cipro£oxacin, a bacteriophage-inducing antimicrobial agent, to mice enhanced the intraintestinal transfer of Stx2 prophage. Therefore, 60 Co irradiation of contaminated foods at ‘low’ doses (e.g. 0.1^0.3 kGy) may provide risks of Stx phage transfer and emergence of new Stxproducing bacteria as well as Stx dissemination, although ‘high’ doses at e.g. 10 kGy completely blocked Stx phage induction and Stx production. In this study, we also investigated inactivation of puri¢ed Stx phage by 60 Co irradiation. Stx phage was more resistant to 60 Co irradiation, compared with STEC. For instance, by 1-kGy irradiation, STEC viable cell counts decreased by 1U109 or more, while PFU of Stx phage decreased by only about 1U101 . However, at 10 kGy or higher, drastic inactivation of Stx phage was observed. In experiments where puri¢ed Stx was irradiated by
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Co, Stx activity was measured using two methods, measurement of cytotoxicity to Vero cells and measurement of in£ammatory cytokine (TNF-K) production in human peripheral blood monocytes, resulting in considerably di¡erent observations among di¡erent assay systems. The cytotoxicity was susceptible to 60 Co irradiation, while the cytokine-inducing activity was resistant to 60 Co irradiation (up to 20 kGy). Stx consists of a single molecule of A subunit and a pentamer of B subunit [1]. The A subunit has N-glycosidase activity and cleaves a single adenine residue of the 28S ribosomal RNA, and the B subunit binds to Gb3 receptor on the cell membrane [1]. The A subunit activity is necessary for both the cytotoxicity and cytokine induction [25,26]. Most probably, Stx is easily destroyed by 60 Co irradiation at 1 kGy or higher, in such a way that the holotoxin (A1B5) dissociates into the A subunit and the B subunit pentamer, losing cytotoxicity, but retaining cytokine induction activity (possibly due to the phagocytized A subunit activity). It is also possible that the holotoxin (A1B5) has a 60 Co irradiation-susceptible domain which is important for cytotoxicity but not for cytokine induction activity. These possibilities are under investigation. In conclusion, 60 Co irradiation at 0.1^0.3 kGy resulted in marked Stx phage induction. The order of susceptibility to 60 Co irradiation was STEC viability, cytotoxic activity of Stx, Stx phage activity and cytokine-inducing activity of Stx. 60 Co irradiation of STEC-contaminated foods at a low dose may lead to Stx phage transfer and emergence of new Stx-producing bacteria (and the dissemination of phage-encoded Stx).
Acknowledgements This work was supported by a Grant-in-Aid for Scienti¢c Research from the Ministry of Education, Culture, Sports, Science and Technology, a grant (97-1) from the Organization for Pharmaceutical Safety and Research (OPSR), and an International Cooperation Grant, Japan.
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[5] American gastroenterological association (1995) Consensus conference statement : Escherichia coli O157:H7 infections-an emerging national health crisis, July 11^13, 1994. Gastroenterology 108, 1923^ 1934. [6] Yamamoto, T., Taneike, I., Tamura, Y., Nishi, J. and Kawakami, K. (2000) A novel hemagglutinin of enterohemorrhagic Escherichia coli O86:HNM associated with hemolytic uremic syndrome in a child. Acta Med. Biol. 48, 147^152. [7] Centers for Disease Control and Prevention (CDC) (1993) Update: multistate outbreak of Escherichia coli O157:H7 infections from hamburgers-western United States, 1992^1993. Morb. Mortal. Wkly. Rep. 42, 258^263. [8] Chapman, P.A., Siddons, C.A., Wright, D.J., Norman, P., Fox, J. and Crick, E. (1993) Cattle as a possible source of verocytotoxinproducing Escherichia coli O157 infections in man. Epidemiol. Infect. 111, 439^447. [9] Dean-Nystrom, E.A., Bosworth, B.T., Cray, W.C. and Moon Jr., H.W. (1997) Pathogenicity of Escherichia coli O157:H7 in the intestines of neonatal calves. Infect. Immun. 65, 1842^1848. [10] O’Brien, A.D., Newland, J.W., Miller, S.F., Holmes, R.K., Smith, H.W. and Formal, S.B. (1984) Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or infantile diarrhea. Science 9, 694^696. [11] Morehouse, K.M. (U.S. Food and Drug Administration) (1998) Report on food irradiation : the treatment of foods with ionizing radiation. [12] World Health Organaization (WHO) (1998) WHO decides : food irradiation safe at any level. Public Health Rep. 113, 6. [13] Matsuyama, A. and Umeda, K. (1983) Sprout inhibition in tubers and bulbs, pp. 159^213. CRC Press, Boca Raton, FL. [14] Kado, C.I. and Liu, S.T. (1981) Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145, 1365^ 1373. [15] Yamamoto, T., Koyama, Y., Matsumoto, M., Sonoda, E., Nakayama, S., Uchimura, M., Paveenkittiporn, W., Tamura, K., Yokota, T. and Echeverria, P. (1992) Localized, aggregative, and di¡use adherence to HeLa cells, plastic, and human small intestines by Escherichia coli isolated from patients with diarrhea. J. Infect. Dis. 166, 1295^ 1310. [16] Nataro, J.P. and Kaper, J.B. (1998) Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11, 142^201. [17] Yutsudo, T., Nakabayashi, N., Hirayama, T. and Takeda, Y. (1987) Puri¢cation and some properties of a Vero toxin from Escherichia coli O157:H7 that is immunologically unrelated to Shiga toxin. Microb. Pathog. 3, 21^30. [18] Kojio, S., Zhang, H., Ohmura, M., Gondaira, F., Kobayashi, N. and Yamamoto, T. (2000) Caspase-3 activation and apoptosis induction coupled with the retrograde transport of Shiga toxin: inhibition by brefeldin A. FEMS Immunol. Med. Microbiol. 29, 275^281. [19] Speirs, J.I., Stavric, S. and Konowalchuk, J. (1977) Assay of Escherichia coli heat-labile enterotoxin with vero cells. Infect. Immun. 16, 617^622. [20] Yamasaki, S., Furutani, M., Ito, K., Igarashi, K., Nishibuchi, M. and Takeda, Y. (1991) Importance of arginine at position 170 of the A subunit of Vero toxin 1 produced by enterohemorrhagic Escherichia coli for toxin activity. Microb. Pathog. 11, 1^9. [21] Zhang, H.M., Ou, Z.L., Gondaira, F., Ohmura, M., Kojio, S. and Yamamoto, T. (2001) Protective e¡ect of anisodamine against Shiga toxin-1: Inhibition of cytokine production and increase in the survival of mice. J. Lab. Clin. Med. 137, 93^100. [22] Matsushiro, A., Sato, K., Miyamoto, H., Yamamura, T. and Honda, T. (1999) Induction of prophages of enterohemorrhagic Escherichia coli O157:H7 with nor£oxacin. J. Bacteriol. 181, 2257^2260. [23] Zhang, X., McDaniel, A.D., Wolf, L.E., Keusch, G.T., Waldor, M.K. and Acheson, D.W.K. (2000) Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice. J. Infect. Dis. 181, 664^670.
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Co irradiation of Shiga toxin (Stx)-producing Escherichia coli induces Stx phage
Tatsuo Yamamoto , Seiichi Kojio, Ikue Taneike, Saori Nakagawa, Nobuhiro Iwakura, Noriko Wakisaka-Saito Division of Bacteriology, Department of Infectious Disease Control and International Medicine, Niigata University Graduate School of Medical and Dental Sciences, 757 Ichibanchou, Asahimachidori, Niigata, Japan Received 22 January 2003 ; received in revised form 10 March 2003; accepted 25 March 2003 First published online 16 April 2003
Abstract Shiga toxin (Stx)-producing Escherichia coli (STEC), an important cause of hemolytic uremic syndrome, was completely killed by 60 Co irradiation at 1Ul03 gray (1 kGy) or higher. However, a low dose of irradiation (0.1^0.3 kGy) markedly induced Stx phage from STEC. Stx production was observed in parallel to the phage induction. Inactivation of Stx phage required a higher irradiation dose than that for bacterial killing. Regarding Stx, cytotoxicity was susceptible to irradiation, but cytokine induction activity was more resistant than Stx phage. The findings suggest that (1) although 60 Co irradiation is an effective means to kill the bacteria, it does induce Stx phage at a lower irradiation dose, with a risk of Stx phage transfer and emergence of new Stx-producing strains, and (2) irradiation differentially inactivates some activities of Stx. 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords :
60
Co irradiation ; Shiga toxin-producing Escherichia coli ; Shiga toxin phage; Shiga toxin; Induction; Inactivation
1. Introduction Shiga toxin (Stx)-producing Escherichia coli (STEC) continues to be an important food-borne bacterial pathogen threatening public health [1]. In Japan in 1996, large outbreaks of STEC infection occurred with 17 877 people infected and 12 fatalities [2,3]. STEC is generally associated with intestinal symptoms and also with serious systemic complications such as hemolytic uremic syndrome, especially in the young and elderly [4,5]. STEC includes not only serotype O157:H7 (the most frequently isolated serotype) but also some other serotypes (e.g.) O26 and O86 [1,5,6]. The most common source of STEC O157:H7 infection is undercooked ground beef [5,7]. STEC O157:H7 is isolated from cattle, which are considered as a reservoir of STEC O157:H7 [8,9]. STEC elaborates Stx1 and/or Stx2, both of which are encoded by prophages lysogenized in STEC strains [1,10].
Cobalt-60 (60 Co) irradiation has been employed for preservation of some foods [11,12]. In practice, ‘low’ dose, up to 1Ul03 gray (1 kGy), is used to delay physiological processes (such as sprouting of fresh fruits), and ‘high’ dose, greater than 10 kGy, is used for avoidance of o¡-odors of meat, seafood and others (although such doses do not kill bacterial spores) [11]. In Japan, irradiation at 0.06^0.145 kGy ( 6 0.15 kGy) is used to inhibit sprouting of potatoes [13]. It has not been investigated whether 60 Co irradiation results in Stx phage induction. In this study, we investigated the killing of STEC strains belonging to various serotypes, induction of Stx phage and inactivation of puri¢ed Stx phage and Stx activity by 60 Co irradiation.
2. Materials and methods 2.1. Bacterial strains
* Corresponding author. Tel. : +81 (25) 227 2050; Fax : +81 (25) 227 0762. E-mail address : [email protected] (T. Yamamoto).
Sixteen STEC human strains were tested. They included ¢ve O157:H7 strains (two Stx1þ Stx2þ strains and three Stx2þ strains), ¢ve O26:H11 strains (Stx1þ ), two
0378-1097 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00259-3
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O111:HUT (H, untypable) strains (an Stx1þ Stx2þ strain and an Stx1þ strain), one O128:H2 strain (Stx1þ Stx2þ ), one O128:H12 strain (Stx1þ Stx2þ ), one O145:H3 strain (Stx1þ Stx2þ ) and one O86:H3 strain (Stx2þ ) [6]. The strains were isolated from outbreaks in Japan in 1991 or 1996 and from sporadic cases in Japan in 1996 or 1999. Bovine O157:H7 strains used were two Stx1þ Stx2þ strains and three Stx2þ strains; they were isolated from feces. 2.2. Media and bacterial growth Nutrient agar (Eiken, Tokyo, Japan) was used as solid media. Luria^Bertani (LB) broth (Difco Laboratories, Detroit, MI, USA) was used as liquid media, which was inoculated and incubated at 37‡C for 12^18 h with agitation. TC-SMAC (Denka Seiken Co., Tokyo, Japan) was used as a selective agar plate for STEC O157:H7 strains. 2.3.
60
Co irradiation of STEC strains
Bacteria grown in LB broth at 37‡C were suspended in phosphate-bu¡ered saline (PBS, pH 7.4) at a concentration of ca. 1Ul09 colony-forming units (CFU) ml31 . The bacterial suspension in a plastic tube (diameter 9 mm) was irradiated by 60 Co at 1Ul02 to 3Ul03 Gy (1Ul04 to 3Ul05 rad) at room temperature (ca. 15‡C), and viable counts (CFU) were determined on agar plates. In some experiments, small pieces (1 g) of 60 Co (or UV)pre-irradiated beef were immersed in suspensions of STEC (200 Wl of 1Ul09 CFU ml31 ), and subjected to 60 Co irradiation. The small beef pieces were then placed in PBS (20 ml), homogenized, and viable cell counts in the homogenates were determined on TC-SMAC agar plates. 2.4. Plasmid analysis Plasmid DNA in STEC O157:H7 cells (from a 2-ml portion of the irradiated or non-irradiated samples) was analyzed essentially by a previously described method [14] with some modi¢cations [15]. Plasmid DNA was electrophoresed in 0.5% agarose, stained with ethidium bromide and analyzed with an image analyzer. 2.5. Phage induction, phage plaque assay and Stx assay Stx2þ O157:H7 strain (OB1) and Stx2þ O86:H3 strain (1076) were grown to the exponential phase (ca. 5Ul08 CFU ml31 ) in LB broth at 37‡C, and the bacterial cells were suspended in equal volumes of PBS, followed by 60 Co irradiation. The bacterial cells were then centrifuged and resuspended in the same volume of fresh LB broth, followed by incubation for 30^180 min at 37‡C. The resultant bacterial culture was divided into two parts. One part was centrifuged and the supernatant was ¢l-
trated through a membrane with a pore size of 0.45 Wm, and the ¢ltrate was subjected to phage titration using E. coli indicator strain C600. CaCl2 was added at 10 mM. The numbers of phage plaques that developed in LB soft agar (0.5%) plates (plaque-forming units, PFU) were counted. The plaques (at least 20 plaques on each plate) were examined for the Stx2 gene by PCR assay [16] ; the plaques developed were all from Stx2 phage. The remaining part was subjected to soni¢cation and centrifugation. The supernatant was ¢ltrated through a membrane with a pore size of 0.45 Wm, two-fold serial dilutions of the supernatant were made. Stx was determined by the reverse passive latex agglutination test using a VT detection kit (Denka Seiken). The Stx titers represented the highest dilution to yield positive results. 2.6. Preparation and
60
Co irradiation of Stx2 phage
Stx2 phages from STEC strains OB1 and l076 were adsorbed onto E. coli C600 cells in LB broth containing 10 mM CaCl2 at a multiplicity of infection of 0.01^0.1 for 20 min at 37‡C, followed by incubation for 8 h at 37‡C in LB soft agar (0.5%) containing 10 mM CaCl2 . Stx2 phages were collected, and puri¢ed through CsCl density gradient centrifugation as described [10]. Puri¢ed Stx phage was suspended in LB broth containing 10 mM CaCl2 and irradiated by 60 Co as above. 2.7. Preparation and
60
Co irradiation of Stx2
Stx2 produced from E. coli strain Tp8 was puri¢ed to homogenity as described previously [17,18]. The Stx2 preparation (0.2 mg protein per ml in PBS) contained no detectable endotoxin (less than 0.05 endotoxin units per ml) contamination as determined by a limulus amebocyte lysate assay. Puri¢ed Stx in PBS (500 ng ml31 ) was irradiated by 60 Co as above. 2.8. Cytotoxicity assay The cytotoxicity of Stx to Vero cells was examined as described previously [19,20]. The cytotoxic titers of the irradiated or non-irradiated puri¢ed Stx2 samples were determined using two-fold serial dilutions. The highest toxin dilution that caused lysis of 50% of the Vero cell monolayer was taken as the titer of each sample. 2.9. Preparation of human peripheral blood monocytes Human mononuclear cells were prepared from peripheral blood of healthy volunteers using Ficoll-Paque (Gibco BRL, Grand Island, NY, USA) gradient centrifugation. Adherent monocytes were then obtained after incubation in 24-well culture plates (A/S Nunc, Roskilde, Denmark) for 60 min at 37‡C in a CO2 incubator with subsequent washing under high pressure [21].
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60
Co irradiation
Origin and serotype of STEC (number)
Humans O157 :H7 (n = 5) O26:H11 (n = 5) O111 :HUT (n = 2) O128 (n = 2) O145 :H3 (n = 1) O86:H3 (n = 1) Bovine O157 :H7 (n = 5)
117
Viable cells (CFU ml31 ) after
60
3U102
1U103
3U103
(6.6 S 5.9)U107 (2.3 S 0.4)U108 (3.6 S 2.0)U107 (4.4 S 4.5)U107 1.8U107 2.8U108
(9.0 S 14.8)U105 (8.5 S 4.1)U106 (1.8 S 2.4)U104 (1.3 S 1.3)U104 6U103 1.5U107
6 1.0 6 1.0 6 1.0 6 1.0 6 1.0 6 1.0
6 1.0 6 1.0 6 1.0 6 1.0 6 1.0 6 1.0
(1.3 S 0.1)U108
(2.6 S 3.5)U106
6 1.0
6 1.0
0
1U10
(9.7 S 1.1)U108 (1.1 S 0.2)U109 (9.1 S 1.1)U108 (1.2 S 0.2)U109 9.9U108 1.2U109 (1.2 S 0.4)U109
2.10. Cytokine assay
Co irradiation at: (Gy)
2
0.3 kGy and 1 kGy, respectively, and was barely detected after 3-kGy irradiation ( 6 4.5%).
The human monocytes (5Ul05 cells ml31 ) prepared as above were stimulated with irradiated or non-irradiated Stx2 (5 ng ml31 ) for 18 h at 37‡C in a CO2 incubator. After incubation, tumor necrosis factor K (TNF-K) in the supernatants was assayed with enzyme-linked immunosorbent assay kits (Genzyme, Cambridge, MA, USA) in accordance with the manufacturer’s instructions.
3.3. Stx phage induction by
60
Co irradiation
An outbreak-derived Stx2þ O157:H7 strain (OB1) and a sporadic case-derived Stx2þ O86:H3 strain (1076) were irradiated by various doses of 60 Co, and the irradiated
3. Results 3.1. Killing of STEC human and bovine strains by 60 Co irradiation STEC human strains including outbreak-derived O157:H7 strains and STEC bovine strains at a concentration of ca. 1Ul09 CFU ml31 in PBS were irradiated by 60 Co at 0.1, 0.3, 1 and 3 kGy, and then viable cell counts in each sample were determined (Table 1). All the STEC strains were killed (the viable counts decreased by 1U109 or more) by 60 Co irradiation at 1 kGy or higher. After irradiation at 0.1 kGy and 0.3 kGy, the viable cell counts decreased by about 1U101 to 1U102 and by about 1U102 to 1U105 , respectively. In beef, a higher irradiation dose was required, and the irradiation dose required to completely kill the STEC O157:H7 strains (isolated from an outbreak as well as from bovine) was 3 kGy (Fig. 1). At 1 kGy, 103 ^104 fold more cells were still alive, compared with those in PBS. 3.2. Destruction of DNA present in STEC by 60 Co irradiation The STEC human and bovine strains had a large plasmid(s) of ca. 90 kbp in size. Those large plasmids present in bacterial cells as a covalently closed circular form were destroyed by 60 Co irradiation ; the covalently closed circular form decreased to 70.5%, 37.7% and 14.6% at 0.1 kGy,
Fig. 1. Inactivation of human and bovine O157:H7 strains of STEC, suspended in PBS or contaminating beef, by 60 Co irradiation. Open symbols represent residual bacterial viable counts (CFU ml31 ) after irradiation at 0.1, 0.3, 1 or 3 kGy for STEC suspended in PBS. Initial viable counts of untreated samples were ca. 1Ul09 CFU ml31 . Closed symbols represent residual bacterial viable counts (CFU ml31 ) after irradiation at 0.1, 0.3, 1 or 3 kGy for STEC contaminating beef. In this case, small pieces of beef (1 g) were mixed with ca. 2Ul08 CFU of STEC, and after irradiation, the small beef pieces were placed in 20 ml of PBS, homogenized, and viable cell counts in the homogenates were determined. Open circle with unbroken line represents data from outbreak-derived human strain OB1 suspended in PBS. Open triangle with broken line represents data from bovine strain V29 suspended in PBS. Closed circle with unbroken line represents data from strain OB1 contaminating beef. Closed triangle with broken line represents data from strain V29 contaminating beef. An asterisk shows that the data are below the detection limit ( 6 1 CFU ml31 ).
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bacterial cells were incubated in a fresh medium for 2 h at 37‡C. The resultant culture supernatants were then examined for Stx phage and Stx. 60 Co irradiation of both strains OB1 and 1076 increased Stx2 phage induction (Fig. 2). The phage induction was most marked at 0.1^0.3 kGy (Fig. 2), with STEC viable counts being decreased to 12.5% to 0.1% (for strain OB1) or 23.3% to 1.3% (for strain 1076). The phage induction reached a level 4.1U102 -fold higher (at 0.1 kGy for strain OB1) or 8.3U103 -fold higher (at 0.3 kGy for strain 1076) than the frequency of spontaneous induction. Stx production was observed in parallel to the phage induction (Fig. 2). Similar Stx production was observed,
Fig. 3. Time course of Stx phage propagation and Stx production after 60 Co irradiation in human O157:H7 and O86:H3 strains of STEC. Bacterial strains used were an outbreak-derived, Stx2þ O157:H7 strain (OB1) in A and a sporadic case-derived, Stx2þ O86:H3 strain (1076) in B. Bacterial cells were suspended in PBS at a concentration of ca. 5Ul08 CFU ml31 , and irradiated at 0.3 kGy for phage induction (at this irradiation, viable counts decreased to 0.1% for strain OB1 and 1.3% for strain 1076). The irradiated cells were then incubated in fresh LB broth at 37‡C for phage propagation and Stx production. Stx2 phage (open circle) and Stx2 (open square) in the culture were periodically examined at the indicated time. An asterisk shows that the data are below the detection limit ( 6 1 PFU ml31 or no detectable Stx2 in the original, undiluted samples). Fig. 2. Stx phage induction and Stx production in 60 Co-irradiated human O157:H7 and O86:H3 strains of STEC. Bacterial strains used were an outbreak-derived, Stx2þ O157:H7 strain (OB1) in A and a sporadic case-derived, Stx2þ O86:H3 strain (1076) in B. Bacterial cells suspended in PBS (ca. 5Ul08 CFU ml31 ) were irradiated at 0.01, 0.03, 0.1, 0.3, 1, 3, 10 or 20 kGy, and each sample was divided into two parts. One part was used for viable cell counting (open triangle). The irradiated cells in the remaining part were incubated in fresh LB broth for 2 h at 37‡C and then Stx2 phage (closed circle) and Stx2 (closed square) in the culture were examined. An asterisk shows that the data are below the detection limit ( 6 1 CFU ml31 , 6 1 PFU ml31 or no detectable Stx2 in the original, undiluted samples).
even when the bacterial cultures were sonicated and the supernatant was examined. The STEC cells, irradiated by 60 Co at 0.3 kGy, were incubated in a fresh medium for various times at 37‡C, and then Stx phage titers and Stx titers were examined (Fig. 3). The Stx phage propagation as well as Stx production nearly plateaued after 120 min (for strain OB1) or 90 min (for strain 1076).
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At a higher irradiation dose, the frequency of phage induction (as well as Stx production) decreased drastically, and no detectable phage induction (or Stx production) was observed at 10 kGy or higher (Fig. 2). 3.4. Inactivation of Stx phage by
60
Co irradiation
Next, Stx2 phages, originating from strains OB1 and 1076, were puri¢ed and irradiated by various doses of 60 Co (Fig. 4). The phages were more resistant to 60 Co irradiation than STEC, and complete inactivation of the phage activity required 10 kGy or higher (Fig. 4). 3.5. Inactivation of the Stx activity by
60
Co irradiation
Puri¢ed Stx2 was irradiated by various doses of 60 Co (Fig. 5). The toxin activity was measured using two methods, measurement of cytotoxicity to Vero cells and measurement of in£ammatory cytokine production from human peripheral blood monocytes. When cytotoxic activity against Vero cells was used as the index, the activity decreased drastically by 212 or more at 1-kGy irradiation. In contrast, the activity for in£ammatory cytokine production in human peripheral blood monocytes was resistant to irradiation, and no signi¢cant decrease was observed at 1 kGy. Even at 20-kGy irradiation, decrease was only about 12%.
Fig. 4. Inactivation of puri¢ed Stx2 phage by 60 Co irradiation. Stx2 phages used originated from an outbreak-derived, Stx2þ O157 :H7 strain OB1 (open circle) and a sporadic case-derived, Stx2þ O86:H3 strain 1076 (closed circle). Phage samples were irradiated at 0.1, 0.3, 1, 3, 10 or 20 kGy, and then PFU were determined by the soft agar method. An asterisk shows that the data are below the detection limit ( 6 1 PFU ml31 ).
Fig. 5. Di¡erential inactivation of biological activities of puri¢ed Stx2 by 60 Co irradiation. Stx2 samples in PBS (500 ng ml31 ) were irradiated at 0.01, 0.03, 0.1, 0.3, 1, 3, 10 or 20 kGy, and then the cytotoxic activity against Vero cells (closed circle) and the activity to induce TNF-K from human peripheral blood monocytes (open circle) were examined. An asterisk shows that the data are below the detection limit (no detectable cytotoxicity in the original, undiluted samples).
4. Discussion In this study, we demonstrated that 60 Co irradiation was useful for killing the outbreak-derived as well as sporadic case-derived STEC strains (regardless of their serotypes). However, we found that 60 Co irradiation markedly induces Stx phages under certain conditions. Phage induction was most marked at 0.1^0.3 kGy, with STEC viable counts being decreased to about 1% (and the STEC 90-kbp plasmid DNA being destroyed by about 50%). Under the condition (at 0.1^0.3 kGy), the Stx phage induction reached levels ca. 103 ^104 -fold higher than the frequency of spontaneous induction. Stx production was observed in parallel to the phage induction. The observation is consistent with the previous notion that Stx prophages are induced with agents that damage DNA or inhibit DNA replication [10,22^24]. Zhang et al. [23] have demonstrated that administration of cipro£oxacin, a bacteriophage-inducing antimicrobial agent, to mice enhanced the intraintestinal transfer of Stx2 prophage. Therefore, 60 Co irradiation of contaminated foods at ‘low’ doses (e.g. 0.1^0.3 kGy) may provide risks of Stx phage transfer and emergence of new Stxproducing bacteria as well as Stx dissemination, although ‘high’ doses at e.g. 10 kGy completely blocked Stx phage induction and Stx production. In this study, we also investigated inactivation of puri¢ed Stx phage by 60 Co irradiation. Stx phage was more resistant to 60 Co irradiation, compared with STEC. For instance, by 1-kGy irradiation, STEC viable cell counts decreased by 1U109 or more, while PFU of Stx phage decreased by only about 1U101 . However, at 10 kGy or higher, drastic inactivation of Stx phage was observed. In experiments where puri¢ed Stx was irradiated by
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60
Co, Stx activity was measured using two methods, measurement of cytotoxicity to Vero cells and measurement of in£ammatory cytokine (TNF-K) production in human peripheral blood monocytes, resulting in considerably di¡erent observations among di¡erent assay systems. The cytotoxicity was susceptible to 60 Co irradiation, while the cytokine-inducing activity was resistant to 60 Co irradiation (up to 20 kGy). Stx consists of a single molecule of A subunit and a pentamer of B subunit [1]. The A subunit has N-glycosidase activity and cleaves a single adenine residue of the 28S ribosomal RNA, and the B subunit binds to Gb3 receptor on the cell membrane [1]. The A subunit activity is necessary for both the cytotoxicity and cytokine induction [25,26]. Most probably, Stx is easily destroyed by 60 Co irradiation at 1 kGy or higher, in such a way that the holotoxin (A1B5) dissociates into the A subunit and the B subunit pentamer, losing cytotoxicity, but retaining cytokine induction activity (possibly due to the phagocytized A subunit activity). It is also possible that the holotoxin (A1B5) has a 60 Co irradiation-susceptible domain which is important for cytotoxicity but not for cytokine induction activity. These possibilities are under investigation. In conclusion, 60 Co irradiation at 0.1^0.3 kGy resulted in marked Stx phage induction. The order of susceptibility to 60 Co irradiation was STEC viability, cytotoxic activity of Stx, Stx phage activity and cytokine-inducing activity of Stx. 60 Co irradiation of STEC-contaminated foods at a low dose may lead to Stx phage transfer and emergence of new Stx-producing bacteria (and the dissemination of phage-encoded Stx).
Acknowledgements This work was supported by a Grant-in-Aid for Scienti¢c Research from the Ministry of Education, Culture, Sports, Science and Technology, a grant (97-1) from the Organization for Pharmaceutical Safety and Research (OPSR), and an International Cooperation Grant, Japan.
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