In vitro Inhibition of the Expression of Escherichia coli 0157:H7 Genes Encoding the Shiga Like Toxins by Antimicrobial Agents:Potential Use in the Treatment
of Human Infections 

Ali Kanbar, MD

Elias Rahal, MS

Ghassan M.Matar, PhD

 

Department of Microbiology and Immunology, Faculty of Medicine, American University of Beirut

Beirut, Lebanon

 

KEY WORDS: Rifampicin, gentamicin, shiga-like toxins

ABSTRACT

We assessed the in vitro effect of rifampicin and gentamicin at both the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) on the expression of shiga-like toxins (SLT I and SLT II) in 7 Escherichia coli O157:H7 strains obtained from outbreaks in the United States. DNA extraction and polymerase chain reaction (PCR)-based detection of stx1 and stx2 genes, encoding, respectively, SLT I and SLT II, were performed on all strains. RNA extraction and RT-PCR of stx1 and stx2 genes were performed on the same strains incubated at 37˚C for 18 hours with and without rifampicin at its MIC of 4 mg/mL. RT-PCR was also performed on the same strains incubated with rifampicin at its MIC for 18 hours followed by a 4-hour incubation with rifampicin at its MBC value (32 mg/mL).Toxins released were detected using the VTE C-RPLA "SEIKEN" kit in the presence and absence of rifampicin or gentamicin at their MIC values. Subsequently, toxin release was measured when E. coli strains were incubated separately, initially with rifampicin or gentamicin, at their MIC of 4 mg/mL for 18 hours followed by a 4-hour incubation at the MBC (rifampicin:32 mg/mL or gentamicin: 8 mg/mL). Control strains incubated directly with rifampicin or gentamicin at MBC values were also performed. Our data have shown that rifampicin at its MIC inhibited the transcription of the genes encoding the shiga-like toxins I and II as shown by a negative RT-PCR of stx1 and stx2. Strains incubated with the MIC of rifampicin followed by incubation with the MBC showed similar transcription inhibition profiles. Control strains incubated directly with the MBC of rifampicin showed a positive RT-PCR. Toxin release was also shown to be decreased when the strains were incubated with the MIC of rifampicin (12-fold decrease for SLT I and 16-fold decrease for SLT II) or that of gentamicin (8-fold decrease for SLT I and II). Similar results were obtained when the strains were incubated first at the MIC of the drug and subsequently at its MBC. However, when the strains were incubated directly with the antibiotic (rifampicin or gentamicin) at its MBC, no decrease in the level of toxins released was detected. These results may denote a possible use of these antibiotics in the treatment of E. coli O157:H7 infections. Additional studies are underway to assess the process in vivo.

INTRODUCTION

Escherichia coli O157: H7 is one of the most notorious pathogens associated with hemorrhagic colitis leading to bloody diarrhea, and it has been correlated with the hemolytic uremic syndrome (HUS), a fatal disease known to be the leading cause of acute renal failure in children1 and an important cause of stroke and central nervous system dysfunction. Infection with E. coli O157:H7 seems to be prevalent in the developed world as well as in the Central African Republic and southern Africa. Transmission is via food, water, and by direct contact from person-to-person.2 The main source of infection is undercooked ground beef, owing to the organism's existence as a colonizer of healthy cattle.3

Infectivity of this organism is quite high; only a low dose is needed to cause human infection.4 The mechanism of infection is rather simple. The bacterium attaches to the mucosa of the distal ileum and colon through its outer membrane protein intimin, encoded by the eaeA gene located on the locus of enterocyte effacement (LEE).5 Attachment leads to the production of typical attaching and effacing (A/E) lesions in the host intestinal mucosa.6 The organism produces one or both shiga-like toxin I (SLT I) and shiga-like toxin II (SLT II), respectively, encoded by stx1 and stx2 genes on lambdoid lysogenic bacteriophages. These toxins inhibit protein translation in the host cell and lead to its death.7-9 Thus, it leads to microvascular damage in the wall of the intestine.10 Once the blood circulation has been compromised, SLTs and other bacterial products like lipopolysaccharide (LPS) gain entrance to the circulation. This increases the levels of inflammatory mediators, disrupts hemostatic systems, and injures several cell types, culminating in a myriad of symptoms called the hemolytic uremic syndrome (HUS). This progression usually takes place in patients at the extremes of age. The disease displays a triad of hemolytic anemia, thrombocytopenia, and renal failure. However, other organs may be affected, including the brain.11 The mortality rate is 5%. Conversely, 5% of those who survive the acute phase progress to severe sequelae such as end-stage renal disease or permanent neurologic damage.1,12

Proven risk factors for progression from colitis to HUS include infection with strains that produce SLT II, which has greater toxicity than SLT I,13 use of an antimotility agent, bloody diarrhea, fever, vomiting, elevated blood leukocyte counts, extremes of age, and female gender.4 Whether the use of antimicrobial agents is a risk factor or not remains debatable.

Treatment of the infection with E. coli O157:H7 is mainly based on rehydration and supportive therapy. Antimicrobial treatment is not currently recommended and may even be harmful.14 In vitro trials have shown that the use of certain antibiotics, mainly the quinolones trimethoprim and furazolidone, appears to augment the production of SLTs from E. coli O157:H7.15 Thus, an agent that is effective in combating the pathogen without exacerbating the disease or triggering detrimental sequelae is needed. This is possible by inhibiting toxin synthesis and release before administration of a bactericidal dose of an antimicrobial agent to avoid unwanted sequelae that can be fatal. Because rifampicin acts on inhibition of gene transcription, we assessed the in vitro effect of rifampicin, at a minimal inhibitory concentration, on transcription inhibition of genes encoding SLT I and SLT II in E. coli O157:H7. Likewise, we assessed the in vitro effect of gentamicin, known inhibitor of protein synthesis at the level of translation, in inhibiting the production of shiga-like toxins. Inhibition of production of these proteins by rifampicin or gentamicin may constitute a first step in an antimicrobial treatment regimen of E. coli 0157:H7 infections.

MATERIALS AND METHODS

E. coli O157:H7 Strains

Seven E. coli O157:H7 strains were obtained from the Centers for Disease Control and Prevention (CDC). They were isolated during outbreaks of hemorrhagic colitis in the United States and all have distinct pulsed field gel electrophoresis profiles.

DNA Extraction and PCR

Total DNA was extracted from the 7 E. coli O157:H7 strains using the GFXTM Genomic Blood DNA Purification kit (AmershamPharmacia Biotech, Piscataway, NJ). PCR was performed on DNA extracts to amplify the stx1 and stx2 genes, in separate reactions, using previously published primers.16 PCR amplification was performed in 100 mL reaction mixtures consisting of 10 mL DNA (2 mg/mL) and 90 mL of the amplification mix containing 16 pmol of each primer, 200 mM concentrations of each deoxynucleoside triphosphate, 10 mL of PCR buffer (Amersham Pharmacia Biotech, Piscataway, NJ) and 2.5 U of Taq DNA polymerase (Amersham Pharmacia Biotech, Piscataway, NJ). A thermal cycler (PTC-100, MJ Research Inc. Watertown, MA) was used for amplification for 35 cycles. Each cycle consisted of denaturation at 95˚C for 15 seconds, primer annealing at 60˚C for 45 seconds and extension at 72˚C for 45 seconds. The cycles were followed by a final extension step at 72˚C for 10 minutes.

Amplicons of the stx 1 (475 Bp) and stx2 (863 Bp) genes were electrophoresed on 1% agarose (Sigma. St. Louis, MO) gel in 1x Tris-borate-EDTA buffer at 117 V for 45 minutes. Ethidium bromide (Sigma. St. Louis, MO) of 1 ug/ml was incorporated into the gel for staining. Amplicons were detected under UV light and photographed with type 667 Polaroid film.

Determination of MIC and MBC of rifampicin and gentamicin for E. coli O157: H7. Rifampicin (Sigma. St. Louis, Mo.) was dissolved in methanol whereas gentamicin (Sigma.St.Louis,Mo) was dissolved in sterile distilled water and both were serially diluted in sterile distilled water in a series of tubes. A calibrated amount of 5x105 CFU of E. coli O157: H7 were added to each tube and the minimum inhibitory concentrations (MIC) of the drugs for the 7 strains were determined according to NCCLS recommendations.17 On each supra MIC tube, a 1:100 dilution was performed and subcultured on a SMAC agar plate. The antimicrobial concentration that induced a 99.99% killing was considered to be the minimal bactericidal concentration (MBC).

RNA Extraction and RT-PCR

RNA was extracted using the Rneasy Mini Kit (QIAGEN Gmbh, Germany) according to manufacturer's specifications. RT-PCR was performed using The Ready-To-Go kit (Amersham Pharmacia Biotech) for c-DNA synthesis according to manufacturer's specifications, primers and PCR conditions as described previously for PCR. Extracted RNA of each strain was used as a control reaction in PCR to rule out DNA contamination in RNA extracts. Amplicons were detected on ethidium bromide-stained gel and photographed as described previously for PCR. 

Determination of The Effect of Rifampicin on RT-PCR

Determining the lowest concentration of rifampicin that inhibits the transcription of stx1 and stx2 genes in E. coli O157:H7 was performed as follows: a single bacterial colony from a sorbitol MaConkey Agar plate was inoculated in 5 mL trypticase soy broth (TSB) (either rifampicin-free or containing rifampicin at subMIC concentrations) and incubated at 37˚C for 18 hours. The cells, being in the log phase, were then adjusted to 3.5 McFarland (equivalent to 109 CFU/mL using a Densimat Densitometer for inoculum standardization [BioMerieux, Marcy, L'Etoile, France]) and then subjected to the RNA extraction procedure as described previously. When RNA extraction was to be performed on the strain incubated in MIC concentrations of rifampicin, 109 CFU/mL of the strain was incubated at 37˚C for 18 hours in TSB containing the MIC  concentration of the drug.

The procedure was first performed on a single randomly selected strain. RT-PCR for the two genes was performed on extracted RNA as previously described. The lowest concentration of rifampicin that inhibited transcription of both genes as shown by a negative RT-PCR in one E. coli 0157:H7 strain, was applied to all 7 strains as previously described. RT-PCR was also performed on the same strains when incubated initially with the MIC of rifampicin for 18 hours followed by addition of the drug to the culture to attain the MBC (32 mg/mL) and incubated for 4 more hours.

Reversed Passive Latex Agglutination (RPLA) Assay of SLT I and SLT II

VTEC-RPLA "SEIKEN" kit (Denka Seiken, LTD., Tokyo, Japan) was used as a confirmatory test for SLT I and II release inhibition, by E. coli O157:H7 strains incubated in the presence of rifampicin and gentamicin after adjustment to a concentration of 3.5 McFarland. The procedure was performed according to manufacturer's specifications on strains grown for 16 to 18 hours at 37˚C both in rifampicin-free TSB and in TSB cultures at a concentration of 4 mg/mL of rifampicin. That concentration inhibited mRNA transcription of stx1 and stx2 genes by RT-PCR. The same procedure was performed on strains incubated with gentamicin at an MIC of 4 mg/mL. The second step was to assess the effect of combining the MIC and MBC in the process. The strains were incubated for 16 to 18 hours with the antibiotic at its MIC, followed by the same antibiotic brought to its MBC (32 mg/mL for rifampicin and 8 mg/mL for gentamicin). The cultures were incubated for 4 more hours, followed by detection of the toxins by RPLA.

RESULTS

All strains were positive by PCR for the stx1 and stx2 genes. This step was performed to verify that the genes were not lost due to subculturing, as is known to frequently occur for stx1 and stx2 genes.18 RT-PCR revealed that all 7 strains transcribed stx1 and stx2 genes.

                                 Transcription of stx2 was inhibited in all strains at rifampicin concentrations that were < 0.5 mg/mL, as revealed by a negative RT-PCR. Although there was a marked decrease in band intensity for stx1 at concentrations < 2 mg/mL, complete inhibition of transcription occurred at a concentration of 4 mg/mL of the antimicrobial agent.

Because the concentration of 4 mg/mL of rifampicin was the lowest to potently inhibit transcription of both genes in all 7 strains, this concentration was subsequently used on all strains. Data have shown that transcription of both genes was inhibited at a concentration of 4 mg/mL in all 7 E. coli 0157:H7 strains (Figures 1 and 2). Bacterial strains incubated with MIC followed by MBC also showed a negative RT-PCR for stx 1 and stx2 (data not shown). The MIC of gentamicin was also found to be equal to 4 mg/mL. The MBC values for rifampicin and gentamicin were found to be 32 and 8 mg/mL, respectively.

To confirm data obtained by RT-PCR, the RPLA assay was performed on culture supernatants of E. coli O157:H7 strains grown in both rifampicin-free TSB and in TSB containing 4 mg/mL of rifampicin. This assay has shown an average of 12-fold decrease in titers of SLT I and 16-fold decrease in titers of SLT II in TSB containing rifampicin as compared with SLT I and II controls in drug-free TSB. Similar data were obtained when strains were incubated with the MIC of rifampicin for 18 hours followed by the MBC for 4 hours. However, when the strains were incubated with rifampicin at its MBC of 32 mg/mL directly, no inhibition of toxin production was detected (Figures 3 and 4).19

Because gentamicin acts at the mRNA translation level, toxin inhibition was assessed using the VTEC-RPLA kit only. When the strains were incubated with gentamicin at an MIC of 4 mg/mL, or when incubated with MIC for 18 hours followed by a 4-hour incubation period with an MBC of 8 mg/mL, results showed an 8-fold decrease in the titers of SLT I and SLT II. Similar to the results obtained with rifampicin, there were no decreases in the titers of both toxins when the strains were incubated directly with gentamicin at its MBC (Figures 5 and 6).

Discussion

Our data revealed that the MICs of rifampicin and gentamicin for E. coli O157:H7 is not only capable of inhibiting growth of the bacterium but also of decreasing the release of toxins necessary for virulence and pathogenicity. A rifampicin or gentamicin concentration of 4 mg/mL appears effectively capable of inhibiting the production of SLT I and II. RPLA assay revealed that incubation of the organisms with the MIC of rifampicin or gentamicin showed a marked decrease in the titer of SLTs released when compared with controls not incubated in the presence of the antimicrobial agent.

The inhibited genes are necessary for the infectious phenotype. The role of SLTs extends beyond damaging the microvasculature of the intestinal wall because their cellular receptor, globotriosyl ceramide, is distributed over a plethora of cell types. These include enterocytes, renal, aortic, and brain endothelial cells, glomerular endothelial cells, mesangial cells, proximal and distal renal tubular epithelial cells, monocytes and cells derived from the monocytic cell line, astrocytoma cells, lung epithelial cells, polymorphonuclear cells, erythrocytes, platelets, and B lymphocytes.20 The effect of SLTs on these cells is lethal. Therefore, prevention of disease progression is of ultimate importance. Otherwise, severe sequelae, such as hemolytic uremic syndrome or HUS, may result and consequently complicate the case.

Treatment of this infection at the diarrheal phase with bactericidal doses of an antibiotic may kill the pathogen and lead to lysis of the bacterial cells. In fact, this was proven by our results, because when the strains were incubated directly with the MBC of the antibiotic, the titer of SLT I and SLT II was very high. This can be explained by the fact that bacteria have presynthesized toxins that can be released on cell lysis. The toxins will damage the microvasculature of the colon wall and gain entrance, with other bacterial toxins and antigens, to the host blood stream.11 Likewise, an alternative manner by which antimicrobial treatment could aggravate the disease is by triggering an SOS response within the pathogen can be considered. DNA damage to the bacterium induces expression of the toxin genes on the bacteriophages hosting stx1 or stx2. An SOS response is also followed by bacterial lysis and release of toxins and antigens.15

Our study attempted to separately assay the in vitro inhibitory effect of rifampicin and gentamicin on the production of SLTs. Our goal was to assess the effectiveness of these antibiotics for future implementation in treatment. Rifampicin  exerts its effect by binding the bacterial DNA-dependent RNA polymerase and blocking transcription.21 Although gentamicin binds the 30S ribosomal subunit irreversibly, thus inhibiting protein synthesis, our data have shown that transcription of stx1 occurred at several rifampicin concentrations that inhibited transcription of the stx2 gene. This may reflect differences in affinities of the gene promoters of the toxins to the RNA polymerase, certain structural properties of the enzyme itself, or some other factors. This was not the case for gentamicin, which generated similar inhibition profiles for both SLTs. This may due to the fact that a different mechanism of inhibition is implicated with no differences in affinities to the two toxins.

Monotherapy with rifampicin results in rapid resistance via one-step mutations that alter the subunit structure of the bacterial RNA polymerase.22 Consequently, if rifampicin is to be recommended, then it should not be used alone but incorporated into a course of treatment that involves other drugs like gentamicin. Additional studies are needed to implement a therapeutic strategy for infection caused by this organism.

ACKNOWLEDGMENTS

The authors would like to thank Dr. B. Swaminathan, Food borne and Diarrheal Diseases Branch, Centers for Disease Control and Prevention, for provision of primers and E. coli strains.

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Figure 1. Effect of rifampicin on RT-PCR of the stx1 gene in the 7 E. coli O157: H7 strains. Lane 1: 100 bp ladder; lane 2: negative control; lanes 3, 5, 7, 9, 11, 13, 15: amplicons of the strains when not exposed to rifampicin; lanes 4, 6, 8, 10, 12, 14, 16: disappearance of the amplicons in the strains when incubated in 4 ug/ml of rifampicin.

 

Figure 2. Effect of rifampicin on RT-PCR of the stx2 gene in the 7 E. coli O157: H7 strains. Lane 1: 100 bp ladder; lane 2: negative control; lanes 3, 5, 7, 9, 11, 13, 15: amplicons of the strains when not exposed to rifampicin; lanes 4, 6, 8,10, 12, 14, 16: disappearance of the amplicons in the strains when incubated in 4 ug/ml of rifampicin.

 

Figure 3. Effect of rifampicin on the release of Shiga-Like Toxin I,as detected by the VTEC-RPLA kit.

 

Figure 4. Effect of rifampicin on the release of Shiga-Like Toxin II,as detected by the VTEC-RPLA kit.

 

Figure 5. Effect of gentamicin on the release of Shiga-like toxin I using the VTEC-RPLA kit.

 

Figure 6. Effect of gentamicin on the release of Shiga-like toxin II using the VTEC-RPLA kit.