Antibacterials that are used as growth promoters in animal husbandry can affect the release of Shiga-toxin-2-converting bacteriophages and Shiga toxin 2 from Escherichia coli strains

Bernd Köhler1, Helge Karch1 and Herbert Schmidt1

Institut für Hygiene und Mikrobiologie der Universität Würzburg, Josef-Schneider-Str. 2, D-97080 Würzburg, Germany1

Author for correspondence: Herbert Schmidt. Tel: +49 931 201 3905. Fax: +49 931 201 3445. e-mail: hschmidt{at}hygiene.uni-wuerzburg.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Antibiotics are commonly used as growth promoters in animal husbandry worldwide. This practice has been linked to the emergence of particular antibiotic-resistant bacteria, and is now controversial. In this study, the ability of growth-promoting antibiotics to induce Shiga toxin (Stx)-converting bacteriophages from Stx-producing Escherichia coli (STEC) strains was investigated. Subinhibitory concentrations of the antibacterial growth promoters olaquindox, carbadox, tylosin and monensin were used for induction experiments. The amount of mature Stx-converting phage particles released from induced and non-induced cultures was determined, and the production of Stx was simultaneously measured by ELISA. Whereas the quinoxaline-1,4-dioxide-type antibiotics olaquindox and carbadox enhanced the release of Stx-converting phage particles from STEC cells, tylosin and monensin decreased phage induction. The production of Stx increased or decreased simultaneously with the amount of free phages. The results of this study show that particular antibacterial growth promoters can induce Stx phages. In vivo induction of Stx phages from lysogenic STEC may increase the amount of free phages in the intestine and therefore may contribute to the spread of STEC and development of new STEC pathotypes.

Keywords: antibacterial growth promoters, Shiga-toxin-converting bacteriophages, phage induction, Shiga toxin release, E. coli O157

Abbreviations: AU, absorbance units; MIC, minimal inhibitory concentration; SIC, subinhibitory concentration; STEC, Shiga toxin-producing Escherichia coli; Stx, Shiga toxin


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Shiga toxin (Stx)-producing Escherichia coli (STEC) are able to cause intestinal foodborne diseases such as diarrhoea and haemorrhagic colitis. Moreover, STEC infections can lead to serious and life-threatening extraintestinal complications such as the haemolytic-uraemic syndrome, thrombotic-thrombocytopenic purpura and neurological disorders (Griffin & Tauxe, 1991 ). Stx can damage endothelial cells (Jacewicz et al., 1999 ) as well as tubular cells, which may result in acute renal failure (Williams et al., 1999 ). Thus, secretion of Stx is regarded as the major virulence factor of STEC, which may lead to severe intestinal and systemic symptoms.

Stx-producing strains can be found in at least 100 E. coli serotypes (Strockbine et al., 1998 ). Moreover, several investigations have shown that STEC frequently occur in cattle and other domestic animals such as sheep, goats, pigs and horses (Beutin et al., 1995 ; Trevena et al., 1996 ). In Germany, epidemiological studies monitoring the presence of STEC in cattle revealed that in some herds about 50% of the animals are colonized (Richter et al., 1997 ).

Stx1 and Stx2 are encoded in the genome of lambdoid prophages which are designated Stx-converting bacteriophages (Smith et al., 1983 ; Strockbine et al., 1986 ). Stimuli such as UV light or mitomycin C are known to induce these prophages. As a result of the induction process, bacterial host cells lyse and release mature phage particles. Thus, other bacteria in the surrounding milieu could be infected and lysogenized by such phages. Neely & Friedman (1998) suggested that the stx genes are cotranscribed with the late phage genes and the amount of Stx may therefore be enhanced prior to cell lysis. Grif et al. (1998) reported that the exposure of E. coli O157 isolates to subinhibitory concentrations of antibiotics can lead to either increased or diminished Stx production. Matsushiro et al. (1999) showed that norfloxacin, an antibiotic used in human therapy, is able to stimulate the simultaneous production of Stx and Stx-converting phages.

With regard to animal feeds, the European Community permits the use of subinhibitory concentrations of several antibacterial substances, which have no application in human therapy, as feed additives (Directive 70/524/EEC, modified by Directive 91/249/EEC). Supplementation of animal feeds with such chemicals is considered to have growth-promoting effects on the animals and to stabilize their state of health (Riedel-Caspary, 1986 ). However, the use of antibacterial agents as growth promoters for animals has elicited controversies among experts because it is considered to support the development of antibiotic-resistant bacteria (Witte, 1998 ). In this study, we investigated the impact of antibacterial agents used for growth promotion in animal husbandry on the release of Stx2-converting phages from STEC cells. Furthermore, we double-checked our results by measuring the production of Stx2, which has previously been shown to be linked to the induction of Stx2-converting phages (Mühldorfer et al., 1996 ). Our investigations are necessary to create the basis for experiments to detect or to exclude a linkage between the practice of using antibiotics for growth promotion in animal husbandry and the emergence of new STEC types by infection of particular E. coli strains by free Stx phages.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains.
STEC strains 3538/95 (O157:H7; stx2), 5720/96 (O26:H-; stx2) and 2771/97 (ONT; stx2e) were isolated from patients with diarrhoea or haemolytic-uraemic syndrome. E. coli C600 (933W) harbours the Stx2-converting phage 933W from E. coli O157:H7 strain EDL933. The E. coli K-12 derivatives C600 and DH5{alpha} were used as controls.

Media, chemicals and enzymes.
Bacteria were routinely grown in Luria-Bertani (LB) broth. For growth of lysogenic strains, the medium was supplemented with 5 mM CaCl2. LB soft agar (0·7%, w/v, agar, 5 mM CaCl2) was prepared for plaque assays. Minimal inhibitory concentrations (MICs) of olaquindox, carbadox, tylosin and monensin were determined according to the NCCLS guidelines (National Committee for Clinical Laboratory Standards, 1993 ). Olaquindox, tylosin and monensin were from ICN Biomedicals; mitomycin C and carbadox were from Sigma Aldrich. Stock solutions of antibiotics were prepared as follows. Mitomycin C, 0·5 mg ml-1; carbadox, 512 µg ml-1; olaquindox, 1 mg ml-1 (all in distilled water). For monensin, 5 mg was dissolved in 1 ml 99·8% (v/v) ethanol. Solid tylosin was added to suspensions of lysogenic bacteria to give a final concentration of 512 µg ml-1 or 1024 µg ml-1. AmpliTaq DNA polymerase was purchased from Perkin-Elmer’s Applied Biosystems Division.

Determination of MICs and selection of subinhibitory concentrations used for prophage induction.
Stx2-producing E. coli were grown overnight in Mueller-Hinton (MH) broth. Then 105 c.f.u. samples from these cultures were mixed with serial dilutions of olaquindox, carbadox, tylosin and monensin in MH broth. After overnight incubation at 37 °C, bacterial growth was determined by turbidity and MIC values were determined as the minimal growth-limiting concentrations of the antibacterial agents. Based on these results, subinhibitory concentrations (SICs) used for prophage induction were selected. Final MICs and selected SICs of the antibacterial growth-promoting agents are listed in Table. 1. Mitomycin C is a well-known and potent prophage-inducing agent. Previous assays indicated that high phage titres could be obtained by adding 0·5 µg mitomycin C ml-1 to STEC strains (data not shown). Thus, prophage induction was performed with 0·5 µg mitomycin C ml-1 and results were regarded as positive controls.


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Table 1. MICs and SICs (µg ml-1) used for prophage induction

 
Induction of prophages.
Stx2-producing E. coli strains were cultured overnight at 37 °C on LB agar plates. One colony was suspended in LB broth supplemented with 5 mM CaCl2. Bacteria were grown at 37 °C with rotary shaking at 180 r.p.m. OD600 was measured with a Hitachi U-2000 spectrophotometer. When the OD600 reached values of 0·1–0·3, the number of c.f.u. ml-1 was determined by plating dilutions on LB agar. Simultaneously, induction of prophages was performed by adding SICs of the antibacterial agents mitomycin C (positive control), olaquindox, carbadox, monensin and tylosin to different samples of the bacterial suspensions. Samples were then grown overnight at 37 °C with rotary shaking at 180 r.p.m. Spontaneous induction of prophages and release of Stx2 was determined by overnight cultivation without addition of any agents (negative control). Samples with monensin contained 2·5% (v/v) ethanol. Therefore, controls also contained 2·5% (v/v) ethanol.

Quantification of phages released from induced lysogenic E. coli strains.
The number of vegetative phages released after prophage induction was determined by plaque assays. Induced lysogenic E. coli strains were grown overnight in LB broth at 37 °C with rotary shaking at 180 r.p.m. Liquid cultures were then centrifuged at 4 °C and 2600 g with a Sigma 3K20 centrifuge. Supernatants were filter-sterilized through 0·2 µm pore-size filters (Schleicher & Schuell). Resulting filtrates contained free phage particles and Stx. One aliquot of each sample was immediately frozen at -20 °C and a second aliquot was diluted tenfold from 101 to 107. Samples and their dilutions were mixed with a suspension of E. coli K-12 indicator strain DH5{alpha} (108 c.f.u. ml-1) in LB soft agar, supplemented with 5 mM CaCl2 and poured on LB agar plates. These platings were performed in duplicate. After overnight incubation at 37 °C, the number of plaques formed in the top layer was determined. The frozen sample was used for Stx determination.

Plaque hybridization.
In order to distinguish plaques resulting from Stx2- or Stx2e-converting phages and plaques resulting from other phages, Southern blot hybridization with a digoxigenin-labelled stxB2- or stxB2e-specific gene probe was performed by transferring phage DNA from plaques to nylon membranes (Bio-Rad) using standard techniques (Sambrook et al., 1989 ). Determination of Stx-positive plaque numbers was performed twice. This method allowed determination of absolute phage titres of Stx2- or Stx2e-converting phages. The absolute phage titre was defined as the number of infectious phages present in 1 ml filtered culture supernatant of an overnight culture as determined by a plaque assay. Dividing the absolute phage titre by the number of bacteria present in the suspension (c.f.u. ml-1) at the time of supplementation with antibacterial agents gave the specific phage titre.

Preparation of digoxigenin-labelled stxB2- and stxB2e-specific gene probes.
Gene probes specific for detection of stxB2 were prepared by performing PCR from E. coli C600 (933W) using primers JS1 (5'-CAT GAA GAA GAT GTT TAT GGC G-3') and JS2 (5'-CTC AGT CAT TAT TAA ACT GCA C-3') (ARK Scientific). Gene probes specific for detection of stxB2e were prepared by performing PCR from E. coli 2771/97 using primers FK1 (5'-CCC GGA TCC AAG AAG ATG TTT ATA G-3') (Schmidt et al., 1999b ) and FK2 (5'-CCC GAA TTC TCA GTT AAA CTT CAC C-3') (Gunzer et al., 1992 ) (ARK Scientific). PCR products were digoxigenin-labelled by a second PCR performed with the same primers and digoxigenin-11-UTP.

Detection and determination of Stx2.
Stx2 was detected and determined by double-antibody (sandwich) ELISA using the Alexon-Trend LMD E. coli Verotoxin Microwell ELISA VERO-35. Absorbance measurements were performed bichromatically at 450/620 nm with a Bio Flow Multiskan MCC/340 ELISA reader. Duplicate samples were run in parallel for each dilution. The resulting absorbance units (AU) were proportional to the amounts of released Stx2 in the samples. To determine the specific Stx2 concentrations, the absolute absorbance values were divided by the number of bacteria (c.f.u. ml-1) present in the suspensions at the time of supplementation with antibacterial agents.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The antibacterial agents tested in this study for their effects on the release of Stx-converting phages in vitro, namely olaquindox, carbadox, tylosin and monensin, are all permitted within the European Community (Directive 70/524/EEC, modified by Directive 91/249/EEC) as feed supplements. Four distinct bacterial strains were included in our studies: E. coli laboratory strain C600 harbouring the Stx2-converting phage 933W, and three clinical STEC isolates with differences in serotype and Stx genotype.

Determination of the MICs of the antibacterial growth promoters
Previous assays (data not shown) indicated that the best effects on the induction of Stx phages were obtained when growth of the lysogenic host strains was inhibited but not completely blocked. We first determined the MIC of each of the chemicals for all strains investigated (Table 1). Based on these results we chose the SICs; these were half the MIC, except for monensin (Table 1). The MIC for monensin was 2048 µg ml-1 for C600 (933W) and exceeded 2048 µg ml-1 for the clinical isolates. Thus, we chose a SIC of 128 µg monensin ml-1 for all tested strains.

Effect of growth promoters on the release of Stx phages
To study the effects of antibacterials used as growth promoters on the induction of Stx phages, we determined the number of Stx-phage-specific plaques after induction by plaque hybridization with stx-specific gene probes. The results of these plaque hybridizations are shown as absolute phage titres in Table 2.


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Table 2. Absolute titres of Stx phages (p.f.u. ml-1) and Stx2 concentrations (AU ml-1) after treatment with SICs of the antibacterial growth promoters and without adding any agents (control)

 
After supplementation of the culture medium with the SIC of olaquindox, all STEC strains showed an increased release of Stx2-converting phages (Tables 2 and 3). The highest phage titre was obtained with E. coli C600 (933W). The Stx2-converting prophage of the clinical E. coli strain 3538/95 was also markedly induced by olaquindox. For E. coli 5720/96 and E. coli 2771/97, the release of Stx-converting phages was 1·8- to 2·7-fold higher after induction.


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Table 3. Changes in the specific titres of Stx phages and Stx2 concentrations after treatment with SICs of the antibacterial growth promoters

 
All Stx-converting prophages were also inducible with carbadox (Tables 2 and 3). Induction was highest for strains 3538/95 and 2771/97.

In contrast to the results with olaquindox and carbadox, all STEC strains exposed to tylosin showed a decreased release of Stx2-converting phages (Tables 2 and 3). This decrease was 50-fold with E. coli 5720/96, 2·9-fold with E. coli 2771/97 and 2·6-fold with E. coli C600 (933W). Although spontaneous phage release occurred with E. coli 3538/95 in the absence of antimicrobial treatment, we did not observe infectious Stx2-converting phage particles after treatment of the culture with tylosin.

Exposure of all STEC strains to monensin also resulted in a slight decrease of phage titres (Tables 2 and 3). Compared to non-induced cultures, we determined 4·0-fold fewer Stx2e-converting phages with E. coli 2771/97, 1·5-fold fewer with E. coli 5720/96 and 1·3-fold fewer with E. coli C600 (933W). As observed with tylosin, no infectious Stx2-converting phages were detected after exposure of E. coli 3538/95 to monensin.

We chose mitomycin C, which is often used for experimental induction of Stx-converting phages, to double-check the results of our studies. Induction and therefore increased release of Stx phages occurred with all strains. The increase in specific phage titres ranged from 9·43 x 101 for E. coli strain 3538/95 to 3·88 x 103 for strain 5720/96 (Table 3).

Correlation of Stx production and Stx phage release
Culture supernatants which were used for determination of phage titres were also used for the ELISA measurement of Stx production. The amount of Stx decreased or increased simultaneously with the phage titres for E. coli strains C600 (933W), 3538/95 and 5720/96 (Tables 2 and 3). The Stx2e-harbouring strain 2771/97 could not be investigated with the ELISA method described, since Stx2e is not detectable by the commercial kit.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The observation that supplementation of animal feed with subtherapeutic concentrations of particular antibacterial agents improves feed conversion in food-producing animals was made in the 1940s (Moore et al., 1946 ). Animals receiving antibacterials as growth promoters in their feed gained 4-5% more body weight. As a consequence, supplementing animal feed with low doses of antimicrobial agents has become a common practice in modern agriculture worldwide (Witte, 1998 ). The mechanism of this growth promotion is still not understood. Greife & Berschauer (1988) suggested that bacteria resident in the rumen and intestines are affected by antibiotics and that the symbiotic relationship between bacteria and animal is modified. Furthermore, they reported that antibacterials used as growth promoters are able to reduce the intestinal microflora, which competes with the host for nutrients in the intestinal lumen. They also found that a significant reduction of bacterial density on the intestinal epithelium by antibacterial growth promoters contributed to a more efficient absorption of nutrients by the animal. In addition, the quinoxaline-1,4-dioxide-type antibiotics, carbadox and olaquindox, have anabolic effects on the protein biosynthesis of their host. This effect may be attributed to stimulation of the activity of proteases located at the surface of epithelial cells. Most of the antibiotics used as growth promoters are not absorbed by the intestine and act on Gram-positive bacteria. Only carbadox and olaquindox are absorbed and inhibit growth of both Gram-positive and Gram-negative bacteria.

STEC are involved in large outbreaks and sporadic cases of haemorrhagic colitis and haemolytic-uraemic syndrome worldwide. Following their first description in 1982 (CDC, 1982 ), mainly STEC of serotype O157:H7 were considered to be a serious health risk and were held responsible for the largest outbreaks in the USA and Japan (Ostroff et al., 1990 ; Watanabe et al., 1996 ). More recent reports have described the emergence of new STEC variants (Pierard et al., 1998 ; Schmidt et al., 1999a ). Variations occurred as regards the serotype as well as the composition and genetic structure of virulence determinants (Brunder et al., 1999 ). Although horizontal gene transfer is thought to be responsible for such alterations, this has so far been poorly investigated in STEC strains. All known virulence determinants of STEC are located on mobile genetic elements, which are thought to be acquired by the mechanism mentioned above. However, environmental conditions for mobilization of such genetic elements in vivo have been poorly investigated.

STEC are exposed to various antibacterials in their main reservoir, the food-producing animal. This is the case when domestic animals are treated with antibiotics for prophylaxis, chemotherapy and growth promotion. The four growth-promoting antibiotics investigated in our in vitro study had different effects on the induction of STEC phages. Whereas olaquindox and carbadox strongly enhanced Stx-phage release in all strains, this effect was not observed with tylosin and monensin. The inducing capability of the growth promoters was different from strain to strain. Interestingly, in E. coli O157:H7 strain 3538/96 and E. coli C600 (933W), the amplification of Stx production after treatment with olaquindox was similar, whereas the amplification of phage titres obtained with E. coli C600 (933W) was 13-fold higher than that obtained with E. coli 3538/96. Due to the present opinion that Stx expression is linked with the expression of the late phage genes (Neely & Friedman, 1998 ) we would expect proportional increase or decrease of Stx and phages. However, phage induction is a complex process dependent on various phage and bacterial host genes. E. coli O157:H7 strain 3538/96 and E. coli laboratory strain C600 (933W) represent a different genetic background and we therefore could also expect differences also in the phage-induction capacity. Since stx genes are known to carry their own promoters, it is also possible that phage induction and stx expression are not strictly coupled.

Carbadox and olaquindox are allowed as supplements in concentrations of 20–50 mg kg-1 and 15–50 mg kg-1, respectively, in feed for piglets (EU Directive 91/248/EEC). Tylosin is allowed in concentrations of 10–40 mg per kg feed (piglets) or 5–20 mg per kg feed (swine). The permitted concentrations for monensin are 10–40 mg kg-1 in feed for cattle. It has been shown for carbadox and olaquindox that, although the concentrations used for growth promotion are subtherapeutic, therapeutic concentrations can be obtained in the upper gastrointestinal tract (de Graaf et al., 1988 ; Spierenburg et al., 1988 ). Moreover, it could be demonstrated that the distribution of substances in the gastrointestinal tract is not homogeneous based on a concentration gradient from the jejunum to the colon. Carbadox was present in the gastrointestinal tract of piglets in a concentration range from 50 to 4 µg per g gut content from the jejunum to the colon, respectively (de Graaf et al., 1988 ). Using olaquindox, concentrations of 0·1–1 µg per g gut content in the lower gastrointestinal tract were obtained, dependent on the doses given in the feed (Spierenburg et al., 1988 ). Since the antibacterial concentrations used in our studies ranged from 0·5 to 8 µg ml-1, we performed our experiments under conditions that may be obtained in vivo.

We showed that the production of Stx correlated with the phage titres: an increase or decrease in the number of phage particles led directly to an increase or decrease, respectively, in the amount of Stx. The induction of lambdoid phages of E. coli in vivo occurs under poor environmental conditions. Starvation, or the influence of aggressive chemicals such as mutagens or UV light, leads to an increased liberation of lambdoid phages. This process is recA-dependent. Since recA is also a component of the SOS response system which repairs DNA damage, we hypothesize that DNA-damaging agents may be generally considered to induce lambdoid phages. The correlation of phage induction and the SOS response system has been demonstrated for STEC phages by Fuchs et al. (1999) , who showed the recA dependency on STEC phage induction. The two quinoxaline-1,4-dioxides, carbadox and olaquindox, are able to damage DNA. We showed that these chemicals also had the capability to induce STEC phages. This observation fits the model described above.

The increased release of Stx phages in vitro after treatment of several STEC with carbadox and olaquindox indicates that these antibacterials have the potential to contribute to an increased amount of free Stx phages in the intestinal environment. As such, phages may act as vectors for the horizontal transfer of stx genes, and resident E. coli strains could be converted to pathogens. Finally, we draw the conclusion that particular antibacterials that are used as growth promoters in animal husbandry may influence the horizontal transfer of virulence genes between bacteria.


   ACKNOWLEDGEMENTS
 
This work was supported by grant Ka 717/3-1 from the Deutsche Forschungsgemeinschaft.


   REFERENCES
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METHODS
RESULTS
DISCUSSION
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Received 24 September 1999; revised 31 December 1999; accepted 25 January 2000.