scholarly journals Efficiency of induction of Shiga-toxin lambdoid prophages in Escherichia coli due to oxidative and antibiotic stress depends on the combination of prophage and the bacterial strain

2019 ◽  
Vol 61 (1) ◽  
pp. 131-140 ◽  
Author(s):  
Michalina Filipiak ◽  
Joanna M. Łoś ◽  
Marcin Łoś
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Laboratory Strain ◽  
Sos Response ◽  
Optical Microscope ◽  
Bacterial Cells ◽  
Prophage Induction ◽  
E Coli ◽  
Lysogenic Culture ◽  
And Oxidative Stress

AbstractIn the study presented here, we tested, how large a fraction of lysogenic culture was undergoing filamentation, which could indicate triggering of the SOS response or SOS-independent prophage induction that is also known to cause cell filamentation. Here, antibiotic stress was triggered by adding mitomycin C and oxidative stress was induced by hydrogen peroxide. Observation of bacterial cells under an optical microscope revealed more filamenting cells for lysogenic Escherichia coli than for strains not carrying a prophage. Moreover, the amount of filamenting cells depended not only on the stress agents used and the type of the prophage, but also on the host. During induction of the 933W prophage, the resulting phage titer and the amount of elongating cells were different when using E. coli O157:H7 EDL933 clinical isolate and the E. coli MG1655 laboratory strain. The amount of filamenting cells correlates well with the observed phage titers.

scholarly journals Sensitization of bacteria to danofloxacin by temperate prophages.

1996 ◽  
Vol 40 (6) ◽  
pp. 1561-1563 ◽  
Author(s):  
S Froshauer ◽  
A M Silvia ◽  
M Chidambaram ◽  
B Sharma ◽  
G M Weinstock
Keyword(s):  
Escherichia Coli ◽  
Sos Response ◽  
Dna Gyrase ◽  
Pasteurella Haemolytica ◽  
Prophage Induction ◽  
E Coli

Danofloxacin (CP-76,136) is in a class of agents that inhibit DNA gyrase and trigger induction of the SOS response and temperate bacteriophages. Killing studies against the bovine pathogen Pasteurella haemolytica demonstrated that danofloxacin exhibits particularly rapid killing kinetics. Here, lysogenic Escherichia coli bearing lambda is found to be more sensitive to danofloxacin than nonlysogenic E. coli. Danofloxacin exposure also induced a prophage in P. haemolytica. The potency of danofloxacin against lysogens in likely enhanced by this prophage induction.

scholarly journals A Validation System for Selection of Bacteriophages against Shiga Toxin-Producing Escherichia coli Contamination

Toxins ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 644
Author(s):  
Agnieszka Necel ◽  
Sylwia Bloch ◽  
Bożena Nejman-Faleńczyk ◽  
Aleksandra Dydecka ◽  
Gracja Topka-Bielecka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Mammalian Cells ◽  
Resistant Bacteria ◽  
Bacterial Cells ◽  
E Coli ◽  
Uremic Syndrome ◽  
Efficiency Of Selection ◽  
Human Infections ◽  
Selection Of

Shiga toxin-producing Escherichia coli (STEC) can cause severe infections in humans, leading to serious diseases and dangerous complications, such as hemolytic-uremic syndrome. Although cattle are a major reservoir of STEC, the most commonly occurring source of human infections are food products (e.g., vegetables) contaminated with cow feces (often due to the use of natural fertilizers in agriculture). Since the use of antibiotics against STEC is controversial, other methods for protection of food against contaminations by these bacteria are required. Here, we propose a validation system for selection of bacteriophages against STEC contamination. As a model system, we have employed a STEC-specific bacteriophage vB_Eco4M-7 and the E. coli O157:H7 strain no. 86-24, bearing Shiga toxin-converting prophage ST2-8624 (Δstx2::cat gfp). When these bacteria were administered on the surface of sliced cucumber (as a model vegetable), significant decrease in number viable E. coli cells was observed after 6 h of incubation. No toxicity of vB_Eco4M-7 against mammalian cells (using the Balb/3T3 cell line as a model) was detected. A rapid decrease of optical density of STEC culture was demonstrated following addition of a vB_Eco4M-7 lysate. However, longer incubation of susceptible bacteria with this bacteriophage resulted in the appearance of phage-resistant cells which predominated in the culture after 24 h incubation. Interestingly, efficiency of selection of bacteria resistant to vB_Eco4M-7 was higher at higher multiplicity of infection (MOI); the highest efficiency was evident at MOI 10, while the lowest occurred at MOI 0.001. A similar phenomenon of selection of the phage-resistant bacteria was also observed in the experiment with the STEC-contaminated cucumber after 24 h incubation with phage lysate. On the other hand, bacteriophage vB_Eco4M-7 could efficiently develop in host bacterial cells, giving plaques at similar efficiency of plating at 37, 25 and 12 °C, indicating that it can destroy STEC cells at the range of temperatures commonly used for vegetable short-term storage. These results indicate that bacteriophage vB_Eco4M-7 may be considered for its use in food protection against STEC contamination; however, caution should be taken due to the phenomenon of the appearance of phage-resistant bacteria.

scholarly journals Human Neutrophils and Their Products Induce Shiga Toxin Production by Enterohemorrhagic Escherichia coli

2001 ◽  
Vol 69 (3) ◽  
pp. 1934-1937 ◽  
Author(s):  
Patrick L. Wagner ◽  
David W. K. Acheson ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Clinical Isolate ◽  
Shiga Toxin ◽  
Toxin Production ◽  
Enterohemorrhagic Escherichia Coli ◽  
Human Neutrophils ◽  
Prophage Induction ◽  
Shiga Toxins ◽  
E Coli

ABSTRACT The Shiga toxins (Stx) are critical virulence factors forEscherichia coli O157:H7 and other serotypes of enterohemorrhagic E. coli (EHEC). These potent toxins are encoded in the genomes of temperate lambdoid bacteriophages. We recently demonstrated that induction of the resident Stx2-encoding prophage in an O157:H7 clinical isolate is required for toxin production by this strain. Since several factors produced by human cells, including hydrogen peroxide (H2O2), are capable of inducing lambdoid prophages, we hypothesized that such molecules might also induce toxin production by EHEC. Here, we studied whether H2O2 and also human neutrophils, an important endogenous source of H2O2, induced Stx2 expression by an EHEC clinical isolate. Both H2O2 and neutrophils were found to augment Stx2 production, raising the possibility that these agents may lead to prophage induction in vivo and thereby contribute to EHEC pathogenesis.

scholarly journals Phenethyl Isothiocyanate Inhibits Shiga Toxin Production in Enterohemorrhagic Escherichia coli by Stringent Response Induction

2014 ◽  
Vol 58 (4) ◽  
pp. 2304-2315 ◽  
Author(s):  
Dariusz Nowicki ◽  
Monika Maciąg-Dorszyńska ◽  
Wioletta Kobiela ◽  
Anna Herman-Antosiewicz ◽  
Alicja Węgrzyn ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Stringent Response ◽  
Enterohemorrhagic Escherichia Coli ◽  
Negative Regulator ◽  
Phenethyl Isothiocyanate ◽  
Prophage Induction ◽  
Content Type ◽  
E Coli ◽  
Stimulation Of

ABSTRACTThe pathogenicity of enterohemorrhagicEscherichia coli(EHEC) depends on production of Shiga toxins, which are encoded bystxgenes located in the genomes of lambdoid prophages. Efficient expression of these genes requires prophage induction and lytic development of phages. Treatment of EHEC infections is problematic due to not only the resistance of various strains to antibiotics but also the fact that many antibiotics cause prophage induction, thus resulting in high-level expression ofstxgenes. Here we report thatE. coligrowth, Shiga toxin-converting phage development, and production of the toxin by EHEC are strongly inhibited by phenethyl isothiocyanate (PEITC). We demonstrate that PEITC induces the stringent response inE. colithat is mediated by massive production of a global regulator, guanosine tetraphosphate (ppGpp). The stringent response induction arises most probably from interactions of PEITC with amino acids and from amino acid deprivation-mediated activation of ppGpp synthesis. In mutants unable to synthesize ppGpp, development of Shiga toxin-converting phages and production of Shiga toxin are significantly enhanced. Therefore, ppGpp, which appears at high levels in bacterial cells after stimulation of its production by PEITC, is a negative regulator of EHEC virulence and at the same time efficiently inhibits bacterial growth. This is in contrast to stimulation of virulence of different bacteria by this nucleotide reported previously by others.

scholarly journals Role for a Phage Promoter in Shiga Toxin 2 Expression from a Pathogenic Escherichia coliStrain

2001 ◽  
Vol 183 (6) ◽  
pp. 2081-2085 ◽  
Author(s):  
Patrick L. Wagner ◽  
Melody N. Neely ◽  
Xiaoping Zhang ◽  
David W. K. Acheson ◽  
Matthew K. Waldor ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Transfer ◽  
Shiga Toxin ◽  
Virulence Gene ◽  
Prophage Induction ◽  
Transcription Terminator ◽  
Shiga Toxins ◽  
E Coli ◽  
Shiga Toxin 2 ◽  
Transcription Complexes

ABSTRACT Shiga toxins (Stxs), encoded by the stxA andstxB genes, are important contributors to the virulence ofEscherichia coli O157:H7 and other Stx-producing E. coli (STEC) strains. The stxA and stxBgenes in STEC strains are located on the genomes of resident prophages of the λ family immediately downstream of the phage late promoters (p R′). The phage-encoded Q proteins modify RNA polymerase initiating transcription at the cognatep R′ promoter which creates transcription complexes that transcend a transcription terminator immediately downstream of p R′ as well as terminator kilobases distal to p R′. To test if this Q-directed processive transcription plays a role instx 2 AB expression, we constructed a mutant prophage in an O157:H7 clinical isolate from whichp R′ and part of Q were deleted but which has an intact pStx, the previously describedstx 2 AB-associated promoter. We report that production of significant levels of Stx2 in this O157:H7 isolate depends on the p R′ promoter. Since transcription initiating at p R′ ultimately requires activation of the phage lytic cascade, expression ofstx 2 AB in STEC depends primarily on prophage induction. By showing this central role for the prophage instx 2 AB expression, our findings contradict the prevailing assumption that phages serve merely as agents for virulence gene transfer.

scholarly journals Transduction of Enteric Escherichia coli Isolates with a Derivative of Shiga Toxin 2-Encoding Bacteriophage φ3538 Isolated from Escherichia coli O157:H7

1999 ◽  
Vol 65 (9) ◽  
pp. 3855-3861 ◽  
Author(s):  
Herbert Schmidt ◽  
Martina Bielaszewska ◽  
Helge Karch
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Laboratory Strain ◽  
Plaque Formation ◽  
Escherichia Coli O157 ◽  
Healthy Individuals ◽  
Wild Type ◽  
E Coli ◽  
Shiga Toxin 2 ◽  
Lysogenic Conversion

ABSTRACT We investigated the ability of a detoxified derivative of a Shiga toxin 2 (Stx2)-encoding bacteriophage to infect and lysogenize entericEscherichia coli strains and to develop infectious progeny from such lysogenized strains. The stx2 gene of the patient E. coli O157:H7 isolate 3538/95 was replaced by the chloramphenicol acetyltransferase (cat) gene from plasmid pACYC184. Phage φ3538(Δstx2 ::cat) was isolated after induction of E. coli O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Stx-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), andE. coli from the physiological stool microflora were infected with φ3538(Δstx2 ::cat), and plaque formation and lysogenic conversion of wild-type E. coli strains were investigated. With the exception of one EIEC strain, none of the E. coli strains supported the formation of plaques when used as indicators for φ3538(Δstx2 ::cat). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC, and 1 of 6 E. coli isolates from the stool microflora of healthy individuals integrated the phage in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of φ3538(Δstx2 ::cat) that formed plaques on a lawn of E. coli laboratory strain C600. The results of our study demonstrate that φ3538(Δstx2 ::cat) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic E. coli and that the lysogens produced infectious phage progeny. Stx-encoding bacteriophages are able to spread stx genes among enteric E. coli strains.

scholarly journals Induction of Shiga Toxin-Converting Prophage in Escherichia coli by High Hydrostatic Pressure

2005 ◽  
Vol 71 (3) ◽  
pp. 1155-1162 ◽  
Author(s):  
Abram Aertsen ◽  
David Faster ◽  
Chris W. Michiels
Keyword(s):  
Escherichia Coli ◽  
Hydrostatic Pressure ◽  
Shiga Toxin ◽  
High Hydrostatic Pressure ◽  
Sos Response ◽  
Resistant Mutant ◽  
Food Preservation ◽  
Bacterial Survival ◽  
E Coli ◽  
Pressure Resistance

ABSTRACT Since high hydrostatic pressure is becoming increasingly important in modern food preservation, its potential effects on microorganisms need to be thoroughly investigated. In this context, mild pressures (<200 MPa) have recently been shown to induce an SOS response in Escherichia coli MG1655. Due to this response, we observed a RecA- and LexA-dependent induction of lambda prophage upon treating E. coli lysogens with sublethal pressures. In this report, we extend this observation to lambdoid Shiga toxin (Stx)-converting bacteriophages in MG1655, which constitute an important virulence trait in Stx-producing E. coli strains (STEC). The window of pressures capable of inducing Stx phages correlated well with the window of bacterial survival. When pressure treatments were conducted in whole milk, which is known to promote bacterial survival, Stx phage induction could be observed at up to 250 MPa in E. coli MG1655 and at up to 300 MPa in a pressure-resistant mutant of this strain. In addition, we found that the intrinsic pressure resistance of two types of Stx phages was very different, with one type surviving relatively well treatments of up to 400 MPa for 15 min at 20°C. Interestingly, and in contrast to UV irradiation or mitomycin C treatment, pressure was not able to induce Stx prophage or an SOS response in several natural Stx-producing STEC isolates.

scholarly journals Characterization of a Shiga Toxin 2e-Converting Bacteriophage from an Escherichia coli Strain of Human Origin

2000 ◽  
Vol 68 (9) ◽  
pp. 4850-4855 ◽  
Author(s):  
Maite Muniesa ◽  
Jürgen Recktenwald ◽  
Martina Bielaszewska ◽  
Helge Karch ◽  
Herbert Schmidt
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Laboratory Strain ◽  
Coli Strain ◽  
Biologically Active ◽  
Trna Genes ◽  
Dependent Manner ◽  
E Coli ◽  
Lysis Cassette ◽  
First Time

ABSTRACT An infectious Shiga toxin (Stx) 2e-converting bacteriophage (φP27) was isolated from Stx2e-producing Escherichia coliONT:H− isolate 2771/97 originating from a patient with diarrhea. The phage could be transduced to E. colilaboratory strain DH5α, and we could show that lysogens were able to produce biologically active toxin in a recA-dependent manner. By DNA sequence analysis of a 6,388-bp HindIII restriction fragment of φP27, we demonstrated that thestx 2e gene was located directly downstream ofileZ and argO tRNA genes. Although no analogue of an antiterminator Q encoding gene was present on this fragment, a lysis cassette comprising two holin genes which are related to the holin genes of Pseudomonas aeruginosa phage φCTX and a gene homologous to the endolysin gene gp19 of phage PS3 were detected. The results of our study demonstrated for the first time that Stx2e can be encoded in the genome of an infectious bacteriophage.

scholarly journals Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

2001 ◽  
Vol 183 (17) ◽  
pp. 5187-5197 ◽  
Author(s):  
Vanessa Sperandio ◽  
Alfredo G. Torres ◽  
Jorge A. Girón ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Mechanism ◽  
Sos Response ◽  
Enterohemorrhagic Escherichia Coli ◽  
Trna Genes ◽  
Wild Type ◽  
E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

One-Step Preparation of Competent E. coli: Transformation and Storage of Bacterial Cells in the Same Solution

2021 ◽  
Vol 2021 (11) ◽  
pp. pdb.prot101212 ◽  
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Escherichia Coli ◽  
Convenient Method ◽  
Plasmid Dna ◽  
Transformation Efficiency ◽  
Bacterial Cells ◽  
E Coli ◽  
One Step ◽  
And Storage

This protocol describes a convenient method for the preparation, use, and storage of competent Escherichia coli. The reported transformation efficiency of this method is ∼5 × 107 transformants/µg of plasmid DNA.

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