Research Toxin Gene Expression by Shiga Toxin-Producing Escherichia coli: the Role of Antibiotics and the Bacterial SOS Response Patrick T. Kimmitt, Colin R. Harwood, Michael R. Barer The Medical School, University of Newcastle, Newcastle upon Tyne, United Kingdom Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through induction of the integrated bacteriophage that encodes the toxin gene. Phage production is linked to induction of the bacterial SOS response, a ubiquitous response to DNA damage. SOS-inducing antimicrobial agents, particularly the quinolones, trimethoprim, and furazolidone, were shown to induce toxin gene expression in studies of their effects on a reporter STEC strain carrying a chromosome- based stx2::lacZ transcriptional fusion. At antimicrobial levels above those required to inhibit bacterial replication, these agents are potent inducers (up to 140-fold) of the transcription of type 2 Shiga toxin genes (stx2); therefore, they should be avoided in treating patients with potential or confirmed STEC infections. Other agents (20 studied) and incubation conditions produced significant but less striking effects on stx2 transcription; positive and negative influences were observed. SOS-mediated induction of toxin synthesis also provides a mechanism that could exacerbate STEC infections and increase dissemination of stx genes. These features and the use of SOS-inducing antibiotics in clinical practice and animal husbandry may account for the recent emergence of STEC disease. The associations between Escherichia coli strains are more closely associated with HUS O157:H7 infection, hemorrhagic colitis, and than are strains that produce only Stx1 (5,6). hemolytic uremic syndrome (HUS) were estab- Because antimicrobial agents may play a role lished in the early 1980s (1,2). Shiga toxin- in the pathogenesis of severe STEC disease, producing E. coli (STEC) strains have since been chemotherapy for STEC infections remains recognized as the cause of both outbreaks and controversial (7,8). The location of stx genes sporadic cases of diarrhea and HUS, involving (predominantly on λ-like bacteriophage genomes thousands of cases and numerous deaths (3). integrated into the chromosome of their host Shiga toxins are key virulence factors in the bacterium) has important implications because pathogenesis of STEC disease (3). The term Shiga the induction of the SOS response, an extensively toxin (Stx) refers to two families of related toxins, characterized genetic regulatory mechanism, Stx/Stx1, which includes the classical Shiga toxin induces high-level expression of previously silent produced by Shigella dysenteriae, and Stx2 (4). bacteriophage genes (9). Stx genes are coexpressed The stx genes carried by STEC strains are, with with genes of the bacteriophage (10,11), and one possible exception (stx2e), encoded on certain quinolones (known to be potent SOS bacteriophage genomes integrated into the inducers) induce increases in toxin (12,13) and bacterial chromosome. Stx2-producing STEC bacteriophage production (13) of two to three orders of magnitude within 2 to 4 hours. The Address for correspondence: M.R. Barer, Department of potential importance of a link between the SOS Microbiology and Immunology, University of Newcastle, The Medical School, Framlington Place, Newcastle upon Tyne, response and prophage induction for Stx1 and NE2 4HH, United Kingdom; Fax: +44 191 222 7736; e-mail: Stx2 expression has been reinforced by recent [email protected]. sequencing and pathogenicity studies (11,14,15). Emerging Infectious Diseases458 Vol. 6, No. 5, September–October 2000 Research We have constructed a genetically modified of prewarmed medium and incubated with derivative of a clinical isolate in which the genes shaking at 37°C to an optical density at 600 nm encoding both elements of the toxin (stx2AB) (OD600nm) of approximately 0.4. The culture was were partially replaced with a lacZ reporter gene. then divided, and test antibiotics were added to The product of this gene, ß-galactosidase, is these aliquots from stock solutions. Aliquots of easily assayed and detected; its expression these cultures (200 µL, 4 replicates) were then reflects the transcriptional activity of the stx2 injected into wells of honeycomb plates (Lab- gene and can be visualized in simple agar plate systems). The Biolink software was used to assays and quantified in biochemical assays. We program the Bioscreen C system to specify have extended our earlier observations on the wavelength, incubation temperature, length of effects of quinolones on reporter expression by experiment, timing of readings, and the rate of this strain to include a wider range of shaking of the cultures. The cultures were antimicrobial agents and the modulating effects incubated in the Bioscreen C system at 37°C with of different environmental conditions. Our continuous shaking, and OD600nm was recorded results show that several agents could increase every 15 min. MICs for selected antibiotics were the amounts of toxin produced and that SOS- determined in the Bioscreen system by using the inducing agents could play an important role in inoculation pattern described above and by the the epidemiology of STEC infections. conventional agar dilution method. Methods Detection of ß-galactosidase Activity An agar plate assay was used to screen the Bacterial Strain and Growth Conditions responses of the reporter strain, PK552 RV31, a strain of E. coli O157:H7 isolated (stx2A::lacZ), in producing ß-galactosidase activ- locally from a patient with hemorrhagic diarrhea, ity in the presence of antibiotics. The assays were was used to construct the reporter strain, performed by using Luria-Bertani (LB) agar PK552(stx2A::lacZ), which contains a copy of the plates containing 20 µg/mL of the chromogenic E. coli lacZ gene transcriptionally fused to the lactose analog X-gal (5-bromo-4-chloro-3-indolyl- promoter region of stx2 on the chromosome. ß-D-galactopyranoside; Sigma, Poole, Dorset, The reporter strain construction, which in- UK). An agar overlay of the PK552 reporter volved allelic exchange with a series of suicide strain was made by mixing 100 µL of an overnight plasmid vectors, will be described elsewhere. In culture with 3 mL of molten LB top agar, cooled to the first step, the entire indigenous lac operon 48°C, and pouring the mixture over the surface of and adjacent lacI gene were deleted to create a an LB agar plate. The plate was incubated with Lac- strain. Then, most of the coding sequence antibiotic discs (see below) placed on the surface, of the stx2A gene was replaced with a under the conditions specified. Expression of promoterless E. coli lacZ gene, resulting in a ß-galactosidase was indicated by the enzymatic transcriptional fusion in which transcription of cleavage of X-gal, resulting in a blue color, the the lacZ reporter gene is controlled by stx2 intensity of which related to the amount of regulatory mechanisms. The structure of the enzyme produced. final construct was confirmed by diagnostic Microaerobic conditions (5% O2, 10% CO2, polymerase chain reaction, Southern blotting, 85% N2) at 37°C were produced in a VAIN and nucleotide sequencing across the lacZ incubator (Don Whitley Scientific, Shipley, insertion points. Yorkshire, UK). Microaerobic conditions at 42°C Bacterial growth in the presence of antibiot- and anaerobic conditions were produced in gas ics was monitored by the Bioscreen C system and jars by using Oxoid gas-generating kits according associated Biolink software (Labsystems, Bas- to the manufacturer’s instructions. ingstoke, UK). This procedure allowed the Antibiotic discs (Oxoid/Unipath, UK) were simultaneous measurement of growth in up to as follows: ofloxacin (5 µg), nalidixic acid 200 tests by recording changes in optical density. (30 µg), cinoxacin (100 µg), enrofloxacin (5 µg): The stx2::lacZ reporter strain PK552 was grown flumequine (30 µg), ciprofloxacin (1 µg), overnight at 37°C in Antibiotic Medium No. 3 perfloxacin (5 µg), norfloxacin (10 µg), amoxycillin/ (Oxoid, Basingstoke, Hampshire, UK). A 2-mL clavulanic acid (20/10 µg), imipenem (10 µg), aliquot of overnight culture was added to 100 mL aztreonam (30 µg), ceftazidime (30 µg), cefotaxime Vol. 6, No. 5, September–October 2000459 Emerging Infectious Diseases Research (30 µg), cefuroxime (5 µg), piperacillin/tazobactam min. ß-galactosidase activity was determined by (10/75 µg), ampicillin (10 µg), cephalexin (30 µg), using the linear portion of the corrected OD420 / chloramphenicol (30 µg), doxycycline (30 µg), time relationship by the Miller formula, adjusted erythromycin (15 µg), trimethoprim (5 µg), for the sample volume (16). Replicate samples (at sulphamethoxazole (25 µg), furazolidone (50 µg), least four in all assays reported) yielded mean amoxicillin (25 µg), novobiocin (30 µg), rifampin values with coefficients of variation <10% in all (25 µg), gentamicin (10 µg), fosfomycin (200 µg) cases. (oral and systemic salts), polymyxin B (300 IU), and metronidazole (50 µg). Results The Bioscreen C system was also used to All the quinolones induced reporter expres- measure total ß-galactosidase activity in whole sion, while only a few of the other agents had this cultures of strain PK552 by orthonitrophenyl-ß- effect (Figure 1). The induction occurred in three D-galactoside (ONPG) assay (16). Aliquots general patterns: a defined zone within the zone (20 µL, 4 replicates) of culture were removed of growth inhibition (quinolones), a defined zone from wells