Psychoactive Drugs Induce the SOS Response and Shiga Toxin Production in Escherichia coli

Several classes of non-antibiotic drugs, including psychoactive drugs, proton-pump inhibitors (PPIs), non-steroidal anti-inflammatory drugs (NSAIDs), and others, appear to have strong antimicrobial properties. We considered whether psychoactive drugs induce the SOS response in E. coli bacteria and, consequently, induce Shiga toxins in Shiga-toxigenic E. coli (STEC). We measured the induction of an SOS response using a recA-lacZ E. coli reporter strain, as RecA is an early, reliable, and quantifiable marker for activation of the SOS stress response pathway. We also measured the production and release of Shiga toxin 2 (Stx2) from a classic E. coli O157:H7 strain, derived from a food-borne outbreak due to spinach. Some, but not all, serotonin selective reuptake inhibitors (SSRIs) and antipsychotic drugs induced an SOS response. The use of SSRIs is widespread and increasing; thus, the use of these antidepressants could account for some cases of hemolytic-uremic syndrome due to STEC and is not attributable to antibiotic administration. SSRIs could have detrimental effects on the normal intestinal microbiome in humans. In addition, as SSRIs are resistant to environmental breakdown, they could have effects on microbial communities, including aquatic ecosystems, long after they have left the human body.

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Nonpathogenic Escherichia coli Can Contribute to the Production of Shiga Toxin

Infection and Immunity ◽

10.1128/iai.71.6.3107-3115.2003 ◽

2003 ◽

Vol 71 (6) ◽

pp. 3107-3115 ◽

Cited By ~ 93

Author(s):

Shantini D. Gamage ◽

Jane E. Strasser ◽

Claudia L. Chalk ◽

Alison A. Weiss

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Gastrointestinal Disease ◽

Intestinal Flora ◽

Gene Promoter ◽

Toxin Production ◽

Lytic Cycle ◽

Resistant Bacteria ◽

E Coli ◽

Shiga Toxin 2

ABSTRACT The food-borne pathogen, Escherichia coli O157:H7, has been associated with gastrointestinal disease and the life-threatening sequela hemolytic uremic syndrome. The genes for the virulence factor, Shiga toxin 2 (Stx2), in E. coli O157:H7 are encoded on a temperate bacteriophage under the regulation of the late gene promoter. Induction of the phage lytic cycle is required for toxin synthesis and release. We investigated the hypothesis that nonpathogenic E. coli could amplify Stx2 production if infected with the toxin-encoding phage. Toxin-encoding phage were incubated with E. coli that were either susceptible or resistant to the phage. The addition of phage to phage-susceptible bacteria resulted in up to 40-fold more toxin than a pure culture of lysogens, whereas the addition of phage to phage-resistant bacteria resulted in significantly reduced levels of toxin. Intestinal E. coli isolates incubated with Shiga toxin-encoding phage produced variable amounts of toxin. Of 37 isolates, 3 produced significantly more toxin than was present in the inoculum, and 1 fecal isolate appeared to inactivate the toxin. Toxin production in the intestine was assessed in a murine model. Fecal toxin recovery was significantly reduced when phage-resistant E. coli was present. These results suggest that the susceptibility of the intestinal flora to the Shiga toxin phage could exert either a protective or an antagonistic influence on the severity of disease by pathogens with phage-encoded Shiga toxin. Toxin production by intestinal flora may represent a novel strategy of pathogenesis.

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Isogenic Lysogens of Diverse Shiga Toxin 2-Encoding Bacteriophages Produce Markedly Different Amounts of Shiga Toxin

Infection and Immunity ◽

10.1128/iai.67.12.6710-6714.1999 ◽

1999 ◽

Vol 67 (12) ◽

pp. 6710-6714 ◽

Cited By ~ 59

Author(s):

Patrick L. Wagner ◽

David W. K. Acheson ◽

Matthew K. Waldor

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Fragment Length ◽

Toxin Production ◽

E Coli ◽

Length Polymorphisms ◽

Shiga Toxin 2 ◽

K 12 ◽

Host Strains ◽

Restriction Fragment Length

ABSTRACT We produced isogenic Escherichia coli K-12 lysogens of seven different Shiga toxin 2 (Stx2)-encoding bacteriophages derived from clinical Shiga toxin-producing E. coli (STEC) isolates of serotypes O157:H7, O145, O111, and O83 to assess the variability among these phages and determine if there were phage-related differences in toxin production. Phage genomic restriction fragment length polymorphisms (RFLP) and superinfection resistance studies revealed significant differences among these phages and allowed the seven phages to be placed into five distinct groups. Experiments revealed striking differences in spontaneous phage and toxin production that were correlated with the groupings derived from the RFLP and resistance studies. These results suggest that the genotype of the Stx2 prophage can influence the level of phage release and toxin expression by host strains and thus may be relevant to STEC pathogenesis.

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Different Effects of Six Antibiotics and Ten Traditional Chinese Medicines on Shiga Toxin Expression byEscherichia coliO157:H7

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/121407 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Mei Ling Chen ◽

Zhao Hao ◽

Yuan Tian ◽

Qi Yao Zhang ◽

Pei Ji Gao ◽

...

Keyword(s):

Shiga Toxin ◽

Mrna Level ◽

Sos Response ◽

Toxin Production ◽

Expression Level ◽

Toxin Gene ◽

Traditional Chinese Medicines ◽

Mrna Levels ◽

Chinese Medicines ◽

E Coli

This study compared the effects of ten types of traditional Chinese medicines (TCMs) and six different antibiotics onE. coliO157:H7 Shiga toxin gene (stx2) mRNA expression level based on real-time PCR and the expression level of Stx toxin using an ELISA quantitative assay. We also compared their effects on the induction of the SOS response. The results clearly indicated that all ten TCMs had negative results in the SOS response induction test, while most TCMs did not increase the levels ofstx2mRNA and the Stx toxin. Some TCMs did increase the mRNA levels of thestx2gene and the Stx toxin level, but their increases were much lower than those caused by antibiotics. With the exception of cefotaxime, the six antibiotics increased the Stx toxin level and increased thestx2gene mRNA level. With the exceptions of cefotaxime and tetracycline, the antibiotics increased the SOS induction response. These results suggest that TCMs may have advantages compared with antibiotics, when treatingE. coliO157:H7; TCMs did not greatly increase Stx toxin production and release.

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Cinnamon Oil Inhibits Shiga Toxin Type 2 Phage Induction and Shiga Toxin Type 2 Production in Escherichia coli O157:H7

Applied and Environmental Microbiology ◽

10.1128/aem.01702-16 ◽

2016 ◽

Vol 82 (22) ◽

pp. 6531-6540 ◽

Cited By ~ 11

Author(s):

Lina Sheng ◽

Barbara Rasco ◽

Mei-Jun Zhu

Keyword(s):

Oxidative Stress ◽

Shiga Toxin ◽

Sos Response ◽

Inhibitory Effect ◽

Cinnamon Oil ◽

Content Type ◽

E Coli ◽

Shiga Toxin 2 ◽

Rna Polyadenylation

ABSTRACTThis study evaluated the inhibitory effect of cinnamon oil againstEscherichia coliO157:H7 Shiga toxin (Stx) production and further explored the underlying mechanisms. The MIC and minimum bactericidal concentration (MBC) of cinnamon oil againstE. coliO157:H7 were 0.025% and 0.05% (vol/vol), respectively. Cinnamon oil significantly reduced Stx2 production and thestx2mRNA expression that is associated with diminished Vero cell cytotoxicity. Consistently, induction of the Stx-converting phage where thestx2gene is located, along with the total number of phages, decreased proportionally to cinnamon oil concentration. In line with decreased Stx2 phage induction, cinnamon oil at 0.75× and 1.0× MIC eliminated RecA, a key mediator of SOS response, polynucleotide phosphorylase (PNPase), and poly(A) polymerase (PAP I), which positively regulate Stx-converting phages, contributing to reduced Stx-converting phage induction and Stx production. Furthermore, cinnamon oil at 0.75× and 1.0× MIC strongly inhibited theqseBCandluxSexpression associated with decreased AI-2 production, a universal quorum sensing signaling molecule. However, the expression of oxidative stress response genesoxyR,soxR, andrpoSwas increased in response to cinnamon oil at 0.25× or 0.5× MIC, which may contribute to stunted bacterial growth and reduced Stx2 phage induction and Stx2 production due to the inhibitory effect of OxyR on prophage activation. Collectively, cinnamon oil inhibits Stx2 production and Stx2 phage induction inE. coliO157:H7 in multiple ways.IMPORTANCEThis study reports the inhibitory effect of cinnamon oil on Shiga toxin 2 phage induction and Shiga toxin 2 production. Subinhibitory concentrations (concentrations below the MIC) of cinnamon oil reduced Stx2 production,stx2mRNA expression, and cytotoxicity on Vero cells. Subinhibitory concentrations of cinnamon oil also dramatically reduced both the Stx2 phage and total phage induction inE. coliO157:H7, which may be due to the suppression of RNA polyadenylation enzyme PNPase at 0.25× to 1.0× MIC and the downregulation of bacterial SOS response key regulator RecA and RNA polyadenylation enzyme PAP I at 0.75× or 1.0× MIC. Cinnamon oil at higher levels (0.75× and 1.0× MIC) eliminated quorum sensing and oxidative stress. Therefore, cinnamon oil has potential applications as a therapeutic to controlE. coliO157:H7 infection through inhibition of bacterial growth and virulence factors.

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The CI Repressors of Shiga Toxin-Converting Prophages Are Involved in Coinfection of Escherichia coli Strains, Which Causes a Down Regulation in the Production of Shiga Toxin 2

Journal of Bacteriology ◽

10.1128/jb.00069-08 ◽

2008 ◽

Vol 190 (13) ◽

pp. 4722-4735 ◽

Cited By ~ 37

Author(s):

R. Serra-Moreno ◽

J. Jofre ◽

M. Muniesa

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Antibiotic Resistance Gene ◽

Vero Cells ◽

Toxin Production ◽

Lytic Cycle ◽

Host Chromosome ◽

E Coli ◽

Shiga Toxin 2 ◽

K 12

ABSTRACT Shiga toxins (Stx) are the main virulence factors associated with a form of Escherichia coli known as Shiga toxin-producing E. coli (STEC). They are encoded in temperate lambdoid phages located on the chromosome of STEC. STEC strains can carry more than one prophage. Consequently, toxin and phage production might be influenced by the presence of more than one Stx prophage on the bacterial chromosome. To examine the effect of the number of prophages on Stx production, we produced E. coli K-12 strains carrying either one Stx2 prophage or two different Stx2 prophages. We used recombinant phages in which an antibiotic resistance gene (aph, cat, or tet) was incorporated in the middle of the Shiga toxin operon. Shiga toxin was quantified by immunoassay and by cytotoxicity assay on Vero cells (50% cytotoxic dose). When two prophages were inserted in the host chromosome, Shiga toxin production and the rate of lytic cycle activation fell. The cI repressor seems to be involved in incorporation of the second prophage. Incorporation and establishment of the lysogenic state of the two prophages, which lowers toxin production, could be regulated by the CI repressors of both prophages operating in trans. Although the sequences of the cI genes of the phages studied differed, the CI protein conformation was conserved. Results indicate that the presence of more than one prophage in the host chromosome could be regarded as a mechanism to allow genetic retention in the cell, by reducing the activation of lytic cycle and hence the pathogenicity of the strains.

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Diversity and Host Range of Shiga Toxin-Encoding Phage

Infection and Immunity ◽

10.1128/iai.72.12.7131-7139.2004 ◽

2004 ◽

Vol 72 (12) ◽

pp. 7131-7139 ◽

Cited By ~ 65

Author(s):

Shantini D. Gamage ◽

Angela K. Patton ◽

James F. Hanson ◽

Alison A. Weiss

Keyword(s):

Shiga Toxin ◽

Foodborne Pathogen ◽

Fold Increase ◽

Toxin Production ◽

Lytic Infection ◽

Antibiotic Resistant ◽

E Coli ◽

Shiga Toxin 2 ◽

Different Strains ◽

Susceptibility And Resistance

ABSTRACT Shiga toxin 2 (Stx2) from the foodborne pathogen Escherichia coli O157:H7 is encoded on a temperate bacteriophage. Toxin-encoding phages from C600::933W and from six clinical E. coli O157:H7 isolates were characterized for PCR polymorphisms, phage morphology, toxin production, and lytic and lysogenic infection profiles on O157 and non-O157 serotype E. coli. The phages were found to be highly variable, and even phages isolated from strains with identical pulsed-field gel electrophoresis profiles differed. Examination of cross-plaquing and lysogeny profiles further substantiated that each phage is distinct; reciprocal patterns of susceptibility and resistance were not observed and it was not possible to define immunity groups. The interaction between Shiga toxin-encoding phage and intestinal E. coli was examined. Lytic infection was assessed by examining Shiga toxin production following overnight incubation with phage. While not common, lytic infection was observed, with a more-than-1,000-fold increase in Stx2 seen in one case, demonstrating that commensal E. coli cells can amplify Shiga toxin if they are susceptible to infection by the Shiga toxin-encoding phages. Antibiotic-resistant derivatives of the Stx2-encoding phages were used to examine lysogeny. Different phages were found to lysogenize different strains of intestinal E. coli. Lysogeny was found to occur more commonly than lytic infection. The presence of a diverse population of Shiga toxin-encoding phages may increase the pathogenic fitness of E. coli O157:H7.

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Detection, enumeration and isolation of strains carrying the stx2 gene from urban sewage

Water Science & Technology ◽

10.2166/wst.2003.0175 ◽

2003 ◽

Vol 47 (3) ◽

pp. 109-116 ◽

Cited By ~ 15

Author(s):

A.R. Blanch ◽

C. García-Aljaro ◽

M. Muniesa ◽

J. Jofre

Keyword(s):

Shiga Toxin ◽

Colony Growth ◽

Coliform Bacteria ◽

Shiga Toxins ◽

E Coli ◽

Urban Sewage ◽

Shiga Toxin 2 ◽

Shiga Toxin Genes ◽

Lateral Transmission ◽

Stx2 Gene

Verotoxigenic Escherichia coli strains have been related with waterborne outbreaks. Besides 0157:H7, several serotypes of E. coli and other enterobacteria have been implicated in outbreaks and reported to carry the shiga toxin genes. Shiga toxins, stx1 and stx2, are important virulence factors of these strains. These genes have been linked to bacteriophages and consequently are susceptible to lateral transmission. To better understand the ecology of these genes a study of the presence of the shiga toxin 2 gene (stx2) among coliform bacteria present in sewage samples was carried out. A procedure based on colony hybridisation was developed for the isolation of enterobacteria carrying this gene. Colony growth on Chromocult® agar was transferred to a membrane and hybridised with a gene specific probe. The procedure allowed detection of about one colony carrying the gene among around 1,000 faecal coliform colonies. The numbers of bacteria carrying the gene in sewage were also estimated by PCR indicating that the numbers of bacteria carrying the stx2 gene were about 1/1,000 faecal coliforms. The detected numbers by both methods were similar. Positive colony hybridisation was detected in four sewage origins. Fifty-two colonies showing positive signal were isolated from the Chromocult® agar plates, confirmed to be stx2 positive by PCR and phenotypically characterised. Results of the characterisation showed certain diversity among the isolates even in isolates from the same sample. Most of these isolates would not have been isolated with the methods regularly used for the isolation of E. coli 0157:H7 strains. The method will allow study of the numbers and characteristics of bacteria carrying the stx2 gene in different water environments and isolate them in order to determine their role in the spread of the gene.

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Replication of plasmids derived from Shiga toxin-converting bacteriophages in starved Escherichia coli

Microbiology ◽

10.1099/mic.0.042820-0 ◽

2011 ◽

Vol 157 (1) ◽

pp. 220-233 ◽

Cited By ~ 10

Author(s):

Bożena Nejman ◽

Beata Nadratowska-Wesołowska ◽

Agnieszka Szalewska-Pałasz ◽

Alicja Węgrzyn ◽

Grzegorz Węgrzyn

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Transcriptional Activation ◽

Stringent Response ◽

Toxin Production ◽

Replication Initiation ◽

Host Cells ◽

Regulation Of Transcription ◽

E Coli ◽

Dna Replication Initiation

The pathogenicity of Shiga toxin-producing Escherichia coli (STEC) depends on the expression of stx genes that are located on lambdoid prophages. Effective toxin production occurs only after prophage induction, and one may presume that replication of the phage genome is important for an increase in the dosage of stx genes, positively influencing their expression. We investigated the replication of plasmids derived from Shiga toxin (Stx)-converting bacteriophages in starved E. coli cells, as starvation conditions may be common in the intestine of infected humans. We found that, unlike plasmids derived from bacteriophage λ, the Shiga toxin phage-derived replicons did not replicate in amino acid-starved relA + and relA − cells (showing the stringent and relaxed responses to starvation, respectively). The presence of the stable fraction of the replication initiator O protein was detected in all tested replicons. However, while ppGpp, the stringent response effector, inhibited the activities of the λ P R promoter and its homologues from Shiga toxin-converting bacteriophages, these promoters, except for λ P R, were only weakly stimulated by the DksA protein. We suggest that this less efficient (relative to λ) positive regulation of transcription responsible for transcriptional activation of the origin contributes to the inhibition of DNA replication initiation of Shiga toxin-converting bacteriophages in starved host cells, even in the absence of ppGpp (as in starved relA − hosts). Possible clinical implications of these results are discussed.

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Recycling of Shiga Toxin 2 Genes in Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

Applied and Environmental Microbiology ◽

10.1128/aem.01906-07 ◽

2007 ◽

Vol 74 (1) ◽

pp. 67-72 ◽

Cited By ~ 44

Author(s):

Alexander Mellmann ◽

Shan Lu ◽

Helge Karch ◽

Jian-guo Xu ◽

Dag Harmsen ◽

...

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Integration Site ◽

Hot Spot ◽

Blot Hybridization ◽

Enterohemorrhagic Escherichia Coli ◽

Biologically Active ◽

E Coli ◽

Shiga Toxin 2

ABSTRACT Using colony blot hybridization with stx 2 and eae probes and agglutination in anti-O157 lipopolysaccharide serum, we isolated stx 2-positive and eae-positive sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) strains from initial stool specimens and stx-negative and eae-positive SF E. coli O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. The stx-negative isolates from each patient shared with the corresponding stx 2-positive isolates fliC H7, non-stx virulence traits, and multilocus sequence types, which indicates that they arose from the stx 2-positive strains by loss of stx 2 during infection. Analysis of the integrity of the yecE gene, a possible stx phage integration site in EHEC O157, in the consecutive stx 2-positive and stx-negative isolates demonstrated that yecE was occupied in stx 2-positive but intact in stx-negative strains. It was possible to infect and lysogenize the stx-negative E. coli O157 strains in vitro using an stx 2-harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of the stx 2-containing phage resulted in the occupation of yecE and production of biologically active Shiga toxin 2. We conclude that the yecE gene in SF E. coli O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and their stx-negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain of stx 2-harboring phages. The ability to recycle stx 2, a critical virulence trait, makes SF E. coli O157:NM strains ephemeral EHEC that can exist as stx-negative variants during certain phases of their life cycle.

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A putative microcin amplifies Shiga toxin 2a production ofEscherichia coliO157:H7

10.1101/646182 ◽

2019 ◽

Author(s):

Hillary M. Figler ◽

Lingzi Xiaoli ◽

Kakolie Banerjee ◽

Maria Hoffmann ◽

Kuan Yao ◽

...

Keyword(s):

Shiga Toxin ◽

Bacterial Infections ◽

Bacterial Species ◽

Treatment Options ◽

Gene Clusters ◽

Toxin Production ◽

Additional Treatment ◽

Proteinase K ◽

Gut Microflora ◽

E Coli

AbstractEscherichia coliO157:H7 is a foodborne pathogen, implicated in various multi-state outbreaks. It encodes Shiga toxin on a prophage, and Shiga toxin production is linked to phage induction. AnE. colistrain, designated 0.1229, was identified that amplified Stx2a production when co-cultured withE. coliO157:H7 strain PA2. Growth of PA2 in 0.1229 cell-free supernatants had a similar effect, even when supernatants were heated to 100°C for 10 min, but not after treatment with Proteinase K. The secreted molecule was shown to use TolC for export and the TonB system for import. The genes sufficient for production of this molecule were localized to a 5.2 kb region of a 12.8 kb plasmid. This region was annotated, identifying hypothetical proteins, a predicted ABC transporter, and a cupin superfamily protein. These genes were identified and shown to be functional in two otherE. colistrains, and bioinformatic analyses identified related gene clusters in similar and distinct bacterial species. These data collectively suggestE. coli0.1229 and otherE. coliproduce a microcin that induces the SOS response in target bacteria. Besides adding to the limited number of microcins known to be produced byE. coli, this study provides an additional mechanism by whichstx2aexpression is increased in response to the gut microflora.ImportanceHow the gut microflora influences the progression of bacterial infections is only beginning to be understood. Antibiotics are counter-indicated forE. coliO157:H7 infections, and therefore treatment options are limited. An increased understanding of how the gut microflora directs O157:H7 virulence gene expression may lead to additional treatment options. This work identifiedE. colithat enhance the production of Shiga toxin by O157:H7, through the secretion of a proposed microcin. This work demonstrates another mechanism by which non-O157E. colistrains may increase Shiga toxin production, and adds to our understanding of microcins, a group of antimicrobials that are less well understood than colicins.

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