Quantification and Evaluation of Infectivity of Shiga Toxin-Encoding Bacteriophages in Beef and Salad

Imamovic, Lejla; Muniesa, Maite

doi:10.1128/aem.02703-10

Cited by 28 publications

(40 citation statements)

References 26 publications

(47 reference statements)

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“…Although we found infectious Stx phages and positive plaques for Stx phages, we did not detect Stx lysogens, supporting previous findings (26). Generation of Stx lysogens may occur at a low frequency, but not all Stx phages are capable of generating lysogens at the same frequency (15) or lysogenizing the same bacterial host strain (32).…”

Section: Discussionsupporting

confidence: 71%

“…Analysis of salad and minced beef samples from the Barcelona area that were suitable for consumption according to European Union norms showed a high prevalence of free Stx phages (26). In several samples, Stx phages proved to be infectious due to their ability to propagate in laboratory E. coli and Shigella strains (26).…”

Section: Discussionmentioning

confidence: 99%

“…Generation of Stx lysogens may occur at a low frequency, but not all Stx phages are capable of generating lysogens at the same frequency (15) or lysogenizing the same bacterial host strain (32). Although the host strains selected here have shown good results for Stx phage detection (15,17,26,32), in previous studies, we found that few or no lysogens are generated, even under the best conditions, using Stx phages recently induced from a lysogenic strain (15). Several explanations can be given to explain why we did not detect Stx lysogens: the small number of Stx phages in the fecal samples, the fact that some Stx phages might not be infectious, the possibility that the strains used are not the optimal hosts, and the presence of other lytic, non-Stx phages.…”

Section: Discussionmentioning

confidence: 99%

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Shiga Toxin 2-Encoding Bacteriophages in Human Fecal Samples from Healthy Individuals

Martínez-Castillo

Quirós

Navarro³

et al. 2013

Appl Environ MicrobiolHas correction 2017-8-1 Has erratum 2017-8-15

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b Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. Previous studies have shown that high densities of free and infectious Stx phages are found in environments polluted with feces and also in food samples. Taken together, these two findings suggest that Stx phages could be excreted through feces, but this has not been tested to date. In this study, we purified Stx phages from 100 fecal samples from 100 healthy individuals showing no enteric symptoms. The phages retrieved from each sample were then quantified by quantitative PCR (qPCR). In total, 62% of the samples carried Stx phages, with an average value of 2.6 ؋ 10 4 Stx phages/g. This result confirms the excretion of free Stx phages by healthy humans. Moreover, the Stx phages from feces were able to propagate in enrichment cultures of stx-negative Escherichia coli (strains C600 and O157:H7) and in Shigella sonnei, indicating that at least a fraction of the Stx phages present were infective. Plaque blot hybridization revealed lysis by Stx phages from feces. Our results confirm the presence of infectious free Stx phages in feces from healthy persons, possibly explaining the environmental prevalence observed in previous studies. It cannot be ruled out, therefore, that some positive stx results obtained during the molecular diagnosis of Shiga toxin-producing Escherichia coli (STEC)-related diseases using stool samples are due to the presence of Stx phages.

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Section: Discussionsupporting

confidence: 71%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Shiga Toxin 2-Encoding Bacteriophages in Human Fecal Samples from Healthy Individuals

Martínez-Castillo

Quirós

Navarro³

et al. 2013

Appl Environ MicrobiolHas correction 2017-8-1 Has erratum 2017-8-15

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“…The fact that stx-negative E. coli (or AEEC) O26:H11 strains could be isolated from stx-positive food samples raises some questions about the diagnostic result since the possibility that these strains are derivatives of STEC that have lost their Stx phage and hence their stx gene during the enrichment procedure or isolation step cannot be excluded. Moreover, Stx phages have also been detected in beef and salad, which may also confound STEC screening methods based on PCR detection of the stx gene in food samples (36).…”

mentioning

confidence: 99%

Heterogeneity in Induction Level, Infection Ability, and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates

Bonanno

Petit

Loukiadis

et al. 2016

Appl Environ Microbiol

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e Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are foodborne pathogens responsible for diarrhea and hemolyticuremic syndrome (HUS). Shiga toxin, the main STEC virulence factor, is encoded by the stx gene located in the genome of a bacteriophage inserted into the bacterial chromosome. The O26:H11 serotype is considered to be the second-most-significant HUScausing serotype worldwide after O157:H7. STEC O26:H11 bacteria and their stx-negative counterparts have been detected in dairy products. They may convert from the one form to the other by loss or acquisition of Stx phages, potentially confounding food microbiological diagnostic methods based on stx gene detection. Here we investigated the diversity and mobility of Stx phages from human and dairy STEC O26:H11 strains. Evaluation of their rate of in vitro induction, occurring either spontaneously or in the presence of mitomycin C, showed that the Stx2 phages were more inducible overall than Stx1 phages. However, no correlation was found between the Stx phage levels produced and the origin of the strains tested or the phage insertion sites. Morphological analysis by electron microscopy showed that Stx phages from STEC O26:H11 displayed various shapes that were unrelated to Stx1 or Stx2 types. Finally, the levels of sensitivity of stx-negative E. coli O26:H11 to six Stx phages differed among the 17 strains tested and our attempts to convert them into STEC were unsuccessful, indicating that their lysogenization was a rare event.

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“…Lactic acid (2%) and peroxyacetic acid containing 1 to 5% H 2 O 2 are used commercially in North America to decontaminate the surfaces of beef carcasses and fresh vegetables (38,43). Stx-phages were found in high numbers on processed beef and salad (44). Because pathogen intervention steps in food processing include acid, oxidative stress, and pressure, it is conceivable that induction of the Stx2-prophage occurs in food processing (18,21,26).…”

Section: Discussionmentioning

confidence: 99%

Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food

Fang

Mercer

McMullen

et al. 2017

Appl Environ Microbiol

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The prophage-encoded Shiga toxin is a major virulence factor in Stxproducing Escherichia coli (STEC). Toxin production and phage production are linked and occur after induction of the RecA-dependent SOS response. However, foodrelated stress and Stx-prophage induction have not been studied at the single-cell level. This study investigated the effects of abiotic environmental stress on stx expression by single-cell quantification of gene expression in STEC O104:H4 Δstx2::gfp:: amp r . In addition, the effect of stress on production of phage particles was determined. The lethality of stressors, including heat, HCl, lactic acid, hydrogen peroxide, and high hydrostatic pressure, was selected to reduce cell counts by 1 to 2 log CFU/ml. The integrity of the bacterial membrane after exposure to stress was measured by propidium iodide (PI). The fluorescent signals of green fluorescent protein (GFP) and PI were quantified by flow cytometry. The mechanism of prophage induction by stress was evaluated by relative gene expression of recA and cell morphology. Acid (pH Ͻ 3.5) and H 2 O 2 (2.5 mM) induced the expression of stx 2 in about 18% and 3% of the population, respectively. The mechanism of prophage induction by acid differs from that of induction by H 2 O 2 . H 2 O 2 induction but not acid induction corresponded to production of infectious phage particles, upregulation of recA, and cell filamentation. Pressure (200 MPa) or heat did not induce the Stx2-encoding prophage (Stx2-prophage). Overall, the quantification method developed in this study allowed investigation of prophage induction and physiological properties at the single-cell level. H 2 O 2 and acids mediate different pathways to induce Stx2-prophage.IMPORTANCE Induction of the Stx-prophage in STEC results in production of phage particles and Stx and thus relates to virulence as well as the transduction of virulence genes. This study developed a method for a detection of the induction of Stxprophages at the single-cell level; membrane permeability and an indication of SOS response to environmental stress were additionally assessed. H 2 O 2 and mitomycin C induced expression of the prophage and activated a SOS response. In contrast, HCl and lactic acid induced the Stx-prophage but not the SOS response. The lifestyle of STEC exposes the organism to intestinal and extraintestinal environments that impose oxidative and acid stress. A more thorough understanding of the influence of food processing-related stressors on Stx-prophage expression thus facilitates control of STEC in food systems by minimizing prophage induction during food production and storage.KEYWORDS single-cell detection, E. coli O104:H4, prophage induction, environmental stress, RecA-independent SOS response, membrane permeability, STEC, Shiga toxin, Shiga toxin-producing E. coli S higa toxin-producing Escherichia coli (STEC) causes the life-threatening hemolyticuremic syndrome (HUS), which is associated with acute renal failure and significant mortality (1, 2). Shiga toxins (Stx) include St...

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Quantification and Evaluation of Infectivity of Shiga Toxin-Encoding Bacteriophages in Beef and Salad

Cited by 28 publications

References 26 publications

Shiga Toxin 2-Encoding Bacteriophages in Human Fecal Samples from Healthy Individuals

Shiga Toxin 2-Encoding Bacteriophages in Human Fecal Samples from Healthy Individuals

Heterogeneity in Induction Level, Infection Ability, and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates

Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food

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