Browsing by Subject "bacteriophages"

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  • Balleste, Elisenda; Blanch, Anicet R.; Mendez, Javier; Sala-Comorera, Laura; Maunula, Leena; Monteiro, Silvia; Farnleitner, Andreas H.; Tiehm, Andreas; Jofre, Joan; Garcia-Aljaro, Cristina (2021)
    The detection of fecal viral pathogens in water is hampered by their great variety and complex analysis. As traditional bacterial indicators are poor viral indicators, there is a need for alternative methods, such as the use of somatic coliphages, which have been included in water safety regulations in recent years. Some researchers have also recommended the use of reference viral pathogens such as noroviruses or other enteric viruses to improve the prediction of fecal viral pollution of human origin. In this work, phages previously tested in microbial source tracking studies were compared with norovirus and adenovirus for their suitability as indicators of human fecal viruses. The phages, namely those infecting human-associated Bacteroides thetaiotaomicron strain GA17 (GA17PH) and porcine-associated Bacteroides strain PG76 (PGPH), and the human-associated crAssphage marker (crAssPH), were evaluated in sewage samples and fecal mixtures obtained from different animals in five European countries, along with norovirus GI + GII (NoV) and human adenovirus (HAdV). GA17PH had an overall sensitivity of >= 83% and the highest specificity (>88%) for human pollution source detection. crAssPH showed the highest sensitivity (100%) and specificity (100%) in northern European countries but a much lower specificity in Spain and Portugal (10 and 30%, respectively), being detected in animal wastewater samples with a high concentration of fecal indicators. The correlations between GA17PH, crAssPH, or the sum of both (BACPH) and HAdV or NoV were higher than between the two human viruses, indicating that bacteriophages are feasible indicators of human viral pathogens of fecal origin and constitute a promising, easy to use and affordable alternative to human viruses for routine water safety monitoring.
  • Hoikkala, Ville; Ravantti, Janne; Diez-Villasenor, Cesar; Tiirola, Marja; Conrad, Rachel A.; McBride, Mark J.; Moineau, Sylvain; Sundberg, Lotta-Riina (2021)
    CRISPR-Cas immune systems adapt to new threats by acquiring new spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition despite variation in spacer content between microbial strains. It has been suggested that such loci may use acquisition machinery from cooccurring CRISPR-Cas systems within the same strain. Here, following infection by a virulent phage with a double-stranded DNA (dsDNA) genome, we observed spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient CRISPR-Cas subtype VI-B system and a complete subtype II-C system. We show that the VI-B locus acquires spacers from both the bacterial and phage genomes, while the newly acquired II-C spacers mainly target the viral genome. Both loci preferably target the terminal end of the phage genome, with priming-like patterns around a preexisting II-C protospacer. Through gene deletion, we show that the RNA-cleaving VI-B system acquires spacers in trans using acquisition machinery from the DNA-cleaving II-C system. Our observations support the concept of cross talk between CRISPR-Cas systems and raise further questions regarding the plasticity of adaptation modules. IMPORTANCE CRISPR-Cas systems are immune systems that protect bacteria and archaea against their viruses, bacteriophages. Immunity is achieved through the acquisition of short DNA fragments from the viral invader's genome. These fragments, called spacers, are integrated into a memory bank on the bacterial genome called the CRISPR array. The spacers allow for the recognition of the same invader upon subsequent infection. Most CRISPR-Cas systems target DNA, but recently, systems that exclusively target RNA have been discovered. RNA-targeting CRISPR-Cas systems often lack genes necessary for spacer acquisition, and it is thus unknown how new spacers are acquired and if they can be acquired from DNA phages. Here, we show that an RNA-targeting system "borrows" acquisition machinery from another CRISPR-Cas locus in the genome. Most new spacers in this locus are unable to target phage mRNA and are therefore likely redundant. Our results reveal collaboration between distinct CRISPR-Cas types and raise further questions on how other CRISPR-Cas loci may cooperate.
  • Mohanraj, Ushanandini (Helsingin yliopisto, 2016)
    The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages are viruses which infect bacteria and are able to produce special proteins involved in bacterial lysis. However, for many bacteriophage-encoded gene products, the function is not known, i.e., hypothetical proteins of unknown function (HPUFs). Screening these proteins likely identifies a rich source of leads that will help in the development of novel antibacterial compounds. The current study presents two phage genomics-based screening approaches to identify phage HPUFs with antibacterial activity. Both screening assays are based on inhibition of bacterial growth when a toxic gene is expression cloned into a plasmid vector. The first approach was a luxAB/luxCDE -based luminescence screening assay. The luxCDE genes encoding the luciferase substrate producing enzymes were integrated into an Escherichia coli strain genome as a transcriptional fusion. Also, a vector carrying the luxAB genes, encoding the luciferase enzyme, and a cloning site for the phage HPUF genes, was constructed. Ligation of a toxic gene into the vector would result in few or rare transformants after electroporation while ligation of a non-toxic gene would result in large number of transformants, and the difference in number of transformants will be reflected in the amount of bioluminescence after electroporation. The proof of concept of the approach was verified using the control genes g150 (a structural, thus a non-toxic gene of phage R1-RT) and regB (a known toxic gene of phage T4). The results demonstrated a significant difference in Relative Luminescence Units (RLU) between the g150 and regB electroporation mixtures. The second screening approach was an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. This assay was tested and optimized with several known control toxic and non-toxic genes. Using the plating assay approach, in the current study, ninety-four R1-RT HPUFs were screened and ten of them showed toxicity in E. coli. In future, the identified toxic HPUFs of R1-RT could be purified and characterized to identify their bacterial targets. Further, both of these screening assays can be used to screen among HPUFs of other phages, and this should allow the discovery of a wide variety of putative inhibitors for the control of current and emerging bacterial pathogens.
  • Kauppinen, Ari; Siponen, Sallamaari; Pitkänen, Tarja; Holmfeldt, Karin; Pursiainen, Anna; Torvinen, Eila; Miettinen, Ilkka T. (2021)
    Bacteriophage control of harmful or pathogenic bacteria has aroused growing interest, largely due to the rise of antibiotic resistance. The objective of this study was to test phages as potential agents for the biocontrol of an opportunistic pathogen Pseudomonas aeruginosa in water. Two P. aeruginosa bacteriophages (vB_PaeM_V523 and vB_PaeM_V524) were isolated from wastewater and characterized physically and functionally. Genomic and morphological characterization showed that both were myoviruses within the Pbunavirus genus. Both had a similar latent period (50-55 min) and burst size (124-134 PFU/infected cell), whereas there was variation in the host range. In addition to these environmental phages, a commercial Pseudomonas phage, JG003 (DSM 19870), was also used in the biocontrol experiments. The biocontrol potential of the three phages in water was tested separately and together as a cocktail against two P. aeruginosa strains; PAO1 and the environmental strain 17V1507. With PAO1, all phages initially reduced the numbers of the bacterial host, with phage V523 being the most efficient (>2.4 log(10) reduction). For the environmental P. aeruginosa strain (17V1507), only the phage JG003 caused a reduction (1.2 log(10)) compared to the control. The cocktail of three phages showed a slightly higher decrease in the level of the hosts compared to the use of individual phages. Although no synergistic effect was observed in the host reduction with the use of the phage cocktail, the cocktail-treated hosts did not appear to acquire resistance as rapidly as hosts treated with a single phage. The results of this study provide a significant step in the development of bacteriophage preparations for the control of pathogens and harmful microbes in water environments.