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Scientific
Publications - Work Done by Microbiology Reader
Emerging Infectious Diseases, Sep-Oct 2000, vol. 6, (5), 11 pp. 458-465 ResearchToxin Gene Expression by Shiga Toxin-Producing Escherichia coli: the Role of Antibiotics and the Bacterial SOS ResponsePatrick T. Kimmitt, Colin R. Harwood, Michael R. Barer
ABSTRACT 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.
INTRODUCTION The associations between Escherichia coli O157:H7 infection, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) were established in the early 1980s (1,2). Shiga toxin-producing E. coli (STEC) strains have since been recognized as the cause of both outbreaks and sporadic cases of diarrhea and HUS, involving thousands of cases and numerous deaths (3). Shiga toxins are key virulence factors in the pathogenesis of STEC disease (3). The term Shiga toxin (Stx) refers to two families of related toxins, Stx/Stx1, which includes the classical Shiga toxin produced by Shigella dysenteriae, and Stx2 (4). The stx genes carried by STEC strains are, with one possible exception (stx2e), encoded on bacteriophage genomes integrated into the bacterial chromosome. Stx2-producing STEC strains are more closely associated with HUS than are strains that produce only Stx1 (5,6). Because antimicrobial agents may play a role in the pathogenesis of severe STEC disease, chemotherapy for STEC infections remains controversial (7,8). The location of stx genes (predominantly on l-like bacteriophage genomes integrated into the chromosome of their host bacterium) has important implications because the induction of the SOS response, an extensively characterized genetic regulatory mechanism, induces high-level expression of previously silent bacteriophage genes (9). Stx genes are coexpressed with genes of the bacteriophage (10,11), and certain quinolones (known to be potent SOS inducers) induce increases in toxin (12,13) and bacteriophage production (13) of two to three orders of magnitude within 2 to 4 hours. The potential importance a link between the SOS response and prophage induction for Stx1 and Stx2 expression has been reinforced by recent sequencing and pathogenicity studies (11,14,15). We have constructed a genetically modified derivative of a clinical isolate in which the genes encoding both elements of the toxin (stx2AB) were partially replaced with a lacZ reporter gene. The product of this gene, b-galactosidase, is easily assayed and detected; its expression reflects the transcriptional activity of the stx2 gene and can be visualized in simple agar plate assays and quantified in biochemical assays. We have extended our earlier observations on the effects of quinolones on reporter expression by this strain to include a wider range of antimicrobial agents and the modulating effects of different environmental conditions. Our results show that several agents could increase the amounts of toxin produced and that SOS-inducing agents could play an important role in the epidemiology of STEC infections.
METHODS Bacterial Strain and Growth ConditionsRV31, a strain of E. coli O157:H7 isolated locally from a patient with hemorrhagic diarrhea, was used to construct the reporter strain, PK552(stx2A::lacZ), which contains a copy of the E. coli lacZ gene transcriptionally fused to the promoter region of stx2 on the chromosome. The reporter strain construction, which involved allelic exchange with a series of suicide plasmid vectors, will be described elsewhere. In the first step, the entire indigenous lac operon and adjacent lacI gene were deleted to create a Lac- strain. Then, most of the coding sequence of the stx2A gene was replaced with a promoterless E. coli lacZ gene, resulting in a transcriptional fusion in which transcription of the lacZ reporter gene is controlled by stx2 regulatory mechanisms. The structure of the final construct was confirmed by diagnostic polymerase chain reaction, Southern blotting, and nucleotide sequencing across the lacZ insertion points. Bacterial growth in the presence of antibiotics was monitored by the Bioscreen C system and associated Biolink software (Labsystems, Basingstoke, UK). This procedure allowed the simultaneous measurement of growth in up to 200 tests by recording changes in optical density. The stx2::lacZ reporter strain PK552 was grown overnight at 37°C in Antibiotic Medium No. 3 (Oxoid, Basingstoke, Hampshire, UK). A 2-mL aliquot of overnight culture was added to 100 mL of prewarmed medium and incubated with shaking at 37°C to an OD600nm of approximately 0.4. The culture was then divided, and test antibiotics were added to these aliquots from stock solutions. Aliquots of these cultures (200 µL, 4 replicates) were then injected into wells of honeycomb plates (Labsystems). The Biolink software was used to program the Bioscreen C system to specify wavelength, incubation temperature, length of experiment, timing of readings, and the rate of shaking of the cultures. The cultures were incubated in the Bioscreen C system at 37°C with continuous shaking, and OD600nm was recorded every 15 min. MICs for selected antibiotics were determined in the Bioscreen system by using the inoculation pattern described above and by the conventional agar dilution method. Detection of b-galactosidase activityAn agar plate assay was used to screen the responses of the reporter strain, PK552 (stx2A::lacZ), in producing b-galactosidase activity in the presence of antibiotics. The assays were performed by using Luria-Bertani (LB) agar plates containing 20 g/mL of the chromogenic lactose analog X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside; Sigma, Poole, Dorset, UK). An agar overlay of the PK552 reporter strain was made by mixing 100 µL of an overnight culture with 3 mL of molten LB top agar, cooled to 48°C, and pouring the mixture over the surface of an LB agar plate. The plate was incubated with antibiotic discs (see below) placed on the surface, under the conditions specified. Expression of -galactosidase was indicated by the enzymatic cleavage of X-gal, resulting in a blue color, the intensity of which related to the amount of enzyme produced. Microaerobic conditions (5% O2, 10% CO2, 85% N2) at 37°C were produced in a VAIN incubator (Don Whitley Scientific, Shipley, Yorkshire, UK). Microaerobic conditions at 42°C and anaerobic conditions were produced in gas jars by using Oxoid gas-generating kits according to the manufacturer's instructions. Antibiotic discs (Oxoid/Unipath, UK) were as follows: ofloxacin (5 µg), nalidixic acid (30 µg), cinoxacin (100 µg), enrofloxacin (5 µg): flumequine (30 µg), ciprofloxacin (1 µg), perfloxacin (5 µg), norfloxacin (10 µg), amoxycillin/ clavulanic acid (20/10 µg), imipenem (10 µg), aztreonam (30 µg), ceftazidime (30 µg), cefotaxime (30 µg), cefuroxime (5 µg), piperacillin/tazobactam (10/75 µg), ampicillin (10 µg), cephalexin (30 µg), chloramphenicol (30 µg), doxycycline (30 µg), erythromycin (15 µg), trimethoprim (5 µg), sulphamethoxazole (25 µg), furazolidone (50 µg), amoxycillin (25 µg), novobiocin (30 µg), rifampin (25 µg), gentamicin (10 µg), fosfomycin (200 µg) (oral and systemic salts), polymyxin B (300 IU), and metronidazole (50 µg). The Bioscreen C system was also used to measure total b-galactosidase activity in whole cultures of strain PK552 by orthonitrophenyl-b-D-galactoside (ONPG) assay (16). Aliquots (20 µL, 4 replicates) of culture were removed from wells of the honeycomb plate and added to a fresh plate. To this, 180 µL of Z buffer (60 mM Na2HPO4 7H2O, 40 mM NaH2PO4 H2O, 10 mM KCl, 1 mM MgSO4, and 50 mM b-mercaptoethanol, pH 7.0) containing 2 mg/mL lysozyme, 0.01% SDS, and 100 µg/mL chloramphenicol was added to the cells. The plate was incubated at 37°C for 30 min to lyse the cells and placed on ice until use. The Bioscreen C system was prewarmed to 28°C, and 40 µL of 4 mg/mL ONPG solution (made up in Z buffer) was added to each well. Plates were incubated in the Bioscreen C system at 28°C for 4 hours, and OD was recorded at both 420 nm and 540 nm every 10 min. b-galactosidase activity was determined by using the linear portion of the corrected OD420 /time relationship by the Miller formula, adjusted for the sample volume (16). Replicate samples (at least four in all assays reported) yielded mean values with coefficients of variation <10% in all cases.
FIGURES
RESULTS All the quinolones induced reporter expression, while only a few of the other agents had this effect (Figure 1). The induction occurred in three general patterns: a defined zone within the zone of growth inhibition (quinolones), a defined zone at the growth/no growth interface (furazolidone), or a diffuse zone within the zone of inhibition (trimethoprim). In addition, the incubation conditions appeared to produce a background level of induction or suppression. Microaerobic conditions and to a lesser extent incubation at 30°C were associated with background induction, while anaerobic conditions and incubation at 42°C had a suppressive effect as determined by the intensities of the blue zones. However, some quinolone-mediated induction was always detectable, even under the most suppressive condition (42°C). Induction by furazolidone and trimethoprim was, in general, similarly enhanced and suppressed, although 42°C abolished all induction and anaerobic conditions did not suppress trimethoprim/sulphamethoxazole induction. As a group, only the agents that inhibit prokaryotic translation failed to induce reporter expression under any condition tested (Table). Like furazolidone, although less intensely, several b-lactam agents induced expression at the growth/no growth interface. Imipenem (a carbapenem) not only failed to induce expression but also inhibited induction by the adjacent monobactam, aztreonam (not shown), at subgrowth-inhibitory levels. We subsequently observed that this suppressive effect was confined to b-lactam-mediated induction (i.e., imipenem showed no suppressive effect against quinolones, trimethoprim, or furazolidone). We also noted an apparent inhibitory effect of clavulanic acid on amoxycillin in the co-amoxyclav combination and a requirement for potentiating conditions (30°C, mAO2 37°C) for detectable induction by novobiocin, polymixin B, and rifampin.
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