Characterizing RecA-Independent Induction of Shiga toxin2-encoding Phages by EDTA Treatment

The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA

Phages in the human body

Bacteriophages, viruses that infect bacteria, have re-emerged as powerful regulators of bacterial populations in natural ecosystems. Phages invade the human body, just as they do other natural environments, to such an extent that they are the most numerous group in the human virome. This was only revealed in recent metagenomic studies, despite the fact that the presence of phages in the human body was reported decades ago. The influence of the presence of phages in humans has yet to be evaluated; but as in marine environments, a clear role in the regulation of bacterial populations could be envisaged, that might have an impact on human health. Moreover, phages are excellent vehicles of genetic transfer, and they contribute to the evolution of bacterial cells in the human body by spreading and acquiring DNA horizontally. The abundance of phages in the human body does not pass unnoticed and the immune system reacts to them, although it is not clear to what extent. Finally, the presence of phages in human samples, which most of the time is not considered, can influence and bias microbiological and molecular results; and, in view of the evidences, some studies suggest that more attention needs to be paid to their interference

The Prophage and Us - Shiga Toxin Phages revisited

The authors first met in 1998 at the University of Würzburg, Germany, at the Institute of Hygiene and Microbiology, in Helge Karch's lab, where Herbert Schmidt worked as a PostDoc and Maite Muniesa visited the lab for a postdoctoral research stay to work on phages encoding Shiga toxin 2e (Stx2e) [1]. Since that time, we have been more or less, as much as our university duties allow, connected by Stx-phage research. Initially described in the early 1980s, Shiga toxin-converting bacteriophages (Stx-phages) have been the subject of numerous publications [ 2,3 ]. The ability to produce Stx, the major pathogenicity factor of enterohemorrhagic E. coli (EHEC), seems to be essentially connected to the location of the Shiga toxin genes (stx) in the genome of lysogenic phages, found always in a similar location within the late transcribed region, and upstream of the lysis and capsid genes [4 - 11]. Stx-phages are double-stranded DNA tailed phages showing a lambdoid or a non-lambdoid genome structure. To the best of our knowledge, stx genes have never been found in other mobile genetic elements other than phages, such as plasmids or pathogenicity islands [12 - 16 ]. Although the genetics and function of Stx-phages have been described in many publications, some basic questions remain still open, for example, (1) why are Stx-phages so successful in terms of evolution and spread among E. coli strains? (2) why do Stx-phages occur mainly in enteropathogenic E. coli strains but not, or only accidentally, in others such as extraintestinal pathogenic E. coli (EXPEC), (3) which advantage do EHEC strains have from the lysogenic state carrying single or multiple Stx-phages

Contribution of cropland to the spread of Shiga toxin phages and the emergence of new Shiga toxin-producing strains

A growing interest in healthy eating has lead to an increase in the consumption of vegetables, associated with a rising number of bacterial outbreaks related to fresh produce. This is the case of the outbreak in Germany, caused by a O104:H4 enteroaggregative E. coli strain lysogenic for a Stx phage. Temperate Stx phages released from their hosts occur as free particles in various environments. This study reports the occurrence of Stx phages in vegetables (lettuce, cucumber, and spinach) and cropland soil samples. Infectious Stx2 phages were found in all samples and many carried also Stx1 phages. Their persistence in vegetables, including germinated sprouts, of Stx phage 933 W and an E. coli C600 (933 W∆stx::gfp-cat) lysogen used as surrogate, showed reductions below 2 log10 units of both microorganisms at 23 °C and 4 °C over 10 days. Higher reductions (up to 3.9 log10) units were observed in cropland soils at both temperatures. Transduction of a recombinant 933 W∆stx::kan phage was observed in all matrices. Protecting against microbial contamination of vegetables is imperative to ensure a safe food chain. Since the emergence of new Stx strains by Stx phage transduction is possible in vegetable matrices, methods aimed at reducing microbial risks in vegetables should not neglect phages

Implications of free Shiga toxin-converting bacteriophages occurring outside bacteria for the evolution and the detection of Shiga toxin-producing Escherichia coli

In this review we highlight recent work that has increased our understanding of the distribution of Shiga toxin-converting phages that can be detected as free phage particles, independently of Shiga toxin-producing bacteria (STEC). Stx phages are a quite diverse group of temperate phages that can be found in their prophage state inserted within the STEC chromosome, but can also be found as phages released from the cell after activation of their lytic cycle. They have been detected in extraintestinal environments such as water polluted with feces from humans or animals, food samples or even in stool samples of healthy individuals. The high persistence of phages to several inactivation conditions makes them suitable candidates for the successful mobilization of stx genes, possibly resulting in the genes reaching a new bacterial genomic background by means of transduction, where ultimately they may be expressed, leading to Stx production. Besides the obvious fact that Stx phages circulating between bacteria can be, and probably are, involved in the emergence of new STEC strains, we review here other possible ways in which free Stx phages could interfere with the detection of STEC in a given sample by current laboratory methods and how to avoid such interference

Bacteriophages in clinical samples can interfere with microbiological diagnostic tools

Bacteriophages are viruses that infect bacteria, and they are found everywhere their bacterial hosts are present, including the human body. To explore the presence of phages in clinical samples, we assessed 65 clinical samples (blood, ascitic fluid, urine, cerebrospinal fluid, and serum). Infectious tailed phages were detected in >45% of ascitic fluid and urine samples. Three examples of phage interference with bacterial isolation were observed. Phages prevented the confluent bacterial growth required for an antibiogram assay when the inoculum was taken from an agar plate containing lysis plaques, but not when taken from a single colony in a phage-free area. In addition, bacteria were isolated directly from ascitic fluid, but not after liquid enrichment culture of the same samples, since phage propagation lysed the bacteria. Lastly, Gram-negative bacilli observed in a urine sample did not grow on agar plates due to the high densities of infectious phages in the sampl

Bacteriophages of Shiga toxin-producing Escherichia coli and their contribution to pathogenicity

Shiga toxins (Stx) of Shiga toxin-producing Escherichia coli (STEC) are generally encoded in the genome of lambdoid bacteriophages, which spend the most time of their life cycle integrated as prophages in specific sites of the bacterial chromosome. Upon spontaneous induction or induction by chemical or physical stimuli, the stx genes are co-transcribed together with the late phase genes of the prophages. After being assembled in the cytoplasm, and after host cell lysis, mature bacteriophage particles are released into the environment, together with Stx. As members of the group of lambdoid phages, Stx phages share many genetic features with the archetypical temperate phage Lambda, but are heterogeneous in their DNA sequences due to frequent recombination events. In addition to Stx phages, the genome of pathogenic STEC bacteria may contain numerous prophages, which are either cryptic or functional. These prophages may carry foreign genes, some of them related to virulence, besides those necessary for the phage life cycle. Since the production of one or more Stx is considered the major pathogenicity factor of STEC, we aim to highlight the new insights on the contribution of Stx phages and other STEC phages to pathogenicity

Bacteriophages as fecal pollution indicators

Bacteriophages are promising tools for the detection of fecal pollution in different environments, and particularly for viral pathogen risk assessment. Having similar morphological and biological characteristics, bacteriophages mimic the fate and transport of enteric viruses. Enteric bacteriophages, especially phages infecting Escherichia coli (coliphages), have been proposed as alternatives or complements to fecal indicator bacteria. Here, we provide a general overview of the potential use of enteric bacteriophages as fecal and viral indicators in different environments, as well as the available methods for their detection and enumeration, and the regulations for their application

Determination of crAssphage in water samples and applicability for tracking human fecal pollution

In recent decades, considerable effort has been devoted to finding microbial source-tracking (MST) markers that are suitable to assess the health risks of faecally polluted waters, with no universal marker reported so far. In this study, the abundance and prevalence of a crAssphage-derived DNA marker in wastewaters of human and animal origins were studied by a new qPCR assay with the ultimate aim of assessing its potential as an MST marker. crAssphage showed up to 106 GC/ml in the sewage samples of human origin, in both the total DNA and the viral DNA fraction. In wastewaters containing animal faecal remains, 39% of the samples were negative for the presence of the crAssphage sequence, while those showing positive results (41% of the samples) were at least 1 log10 unit lower than the samples of human origin. Noteworthy, the log10 values of the ratio (R) crAssphage (GC/ml)/Escherichia coli (CFU/ml) varied significantly depending on the human or animal origin (R > 1.5 for human samples and R < −1.5 for animal wastewater samples. This study opens the way for further research to explore if different specific animal variants of crAssphage exist and whether other zones of the crAssphage genome are better suited to source discrimination

Is Genetic Mobilization Considered When Using Bacteriophages in Antimicrobial Therapy?

The emergence of multi-drug resistant bacteria has undermined our capacity to control bacterial infectious diseases. Measures needed to tackle this problem include controlling the spread of antibiotic resistance, designing new antibiotics, and encouraging the use of alternative therapies. Phage therapy seems to be a feasible alternative to antibiotics, although there are still some concerns and legal issues to overcome before it can be implemented on a large scale. Here we highlight some of those concerns, especially those related to the ability of bacteriophages to transport bacterial DNA and, in particular, antibiotic resistance genes