7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Chair

Developping novel, phage-inspired antibacterial design strategies

Prof. Dr.

Invited Speaker

Invited Talk

Professor

Novel sex-pilus-specific bacteriophages reduce plasmid carriage and transfer in vitro

Phages and microbial defense systems 

Antibiotic resistance genes are often encoded on conjugative plasmids, which facilitate the horizontal transfer and spreading of these genes. Bacteriophages that are targeting the sex pilus of the conjugational apparatus might therefore be useful to remove resistances from bacterial populations by (i) specifically targeting and lysing bacteria that possess a plasmid-encoded sex pilus and (ii) reducing the horizontal transfer of these plasmids.We have identified two novel bacteriophages, ɸ4187/61 and ɸ4187/77, by a spot-assay screening of a collection of phages isolated from wastewater, that are specifically targeting plasmids belonging to Inc P, N and W. Deletion of the transfer genes from the plasmids rendered the bacteria resistant to ɸ4187/61 and ɸ4187/77, indicating that the conjugation apparatus is indeed the receptor of the phages. We show that both bacteriophages reduce the amount of plasmid-harboring bacteria and drive the selection towards plasmid-free bacterial populations in in vitro co-cultivation studies. Furthermore, we show that both bacteriophages also reduce the horizontal transfer of the plasmids in vitro. In addition to that, we have analyzed resistant bacterial isolates after phage treatment and could show that plasmid loss is one of the major phage resistance mechanisms. A second sub-population of phage-resistant bacteria still contained the plasmids. However, the vast majority of plasmid-harboring and phage-resistant bacteria had lost the capability to transfer the plasmid by conjugation, whereas a minority was still able to transfer the plasmid by conjugation, albeit at a strongly reduced frequency.In summary, we present two novel plasmid-specific bacteriophages ɸ4187/61 and ɸ4187/77 that are specifically targeting the conjugational apparatus of plasmids. We show here that these bacteriophages can be used to drive bacterial populations towards plasmid loss and to reduce the horizontal distribution of these plasmids transfer in vitro. Future experiments using suitable animal models will show, if ɸ4187/61 and ɸ4187/77 are also suitable to remove plasmids from bacterial populations in vivo.

Oral Presentation

PhageDive: the new data resource on prokaryotic viruses

Prokaryotic viruses are the most diverse and abundant biological entities on Earth. While the amount of genomic data on viruses is currently growing exponentially, mainly due to the increasing application of metagenomics techniques, these data and those generated with different experimental approaches and molecular techniques often cannot be linked to the physical virus resource. As a result, the comprehensive set of existing data for a given bacteriophage is not accessible so far and different datasets are not linked for integrative analyses. This observation led us to develop PhageDive (phagedive.dsmz.de), a specific database for bacteriophages and archaeal viruses. This comprehensive database gathers all existing data on prokaryotic viruses that are dispersed across multiple sources, like scientific publications, specialized databases or internal files of culture collections. PhageDive allows to link own research data to the existing information through an easy and central access. PhageDive provides fields for various experimental data (lifestyle, lysis kinetics, adsorption kinetics, host range, genomic data, etc.) and all available metadata (e.g., geographical origin, isolation source). Data are standardized employing controlled vocabulary and ontologies. One important feature is to link experimental data to the culture collection number and the repository of the corresponding physical bioresource (virus and prokaryotic host strains). To date, PhageDive covers 1,167 phages from three different world-renowned collections (DSMZ, Canada's Laval collection and the UK's NCTC). An advanced search using all the sections has been created and an interoperable system has been set up with other resources such as NCBI, the Institut Pasteur's Viral host range database (VHRdb) or the DSMZ's BacDive and MediaDive.

Comparison of two host-phage interaction methods in Pseudomonas aeruginosa

Goals: The aim of this work is the systematic comparison of double agar overlay plaque assay (DPA) and plankton killing assay (PKA) on a large phage-strain panel to identify strengths and weaknesses of both methods and to initially conclude which method allows for the best in vivo prediction. In addition, predictions concerning a therapy regime (simultaneous / sequential) will be compared for both methods. Introduction:During phage therapy, usually first a phagogram, similar to an antibiogram, is carried out to determine the suitability of phage(s) of interest. Various methods are described for testing phage efficacy. The two most common are DPA, which tests the host-phage interaction on solid medium, while in PKA host-phage interaction is analysed in liquid. These two methods were compared.Materials &amp; Methods142 Pseudomonas aeruginosa isolates from different clinical backgrounds (urinary tract, rectal, bronchiectasis, COPD, cystic fibrosis, wounds) were used to determine the host range of 27 phages by DPA and PKA. PKA was also performed simultaneously and sequentially on selected strains using 8 strictly lytic phages.Results:The host spectra determined using DPA and PKA differed considerably. For Pakpunavirus, Pbunavirus and Litunavirus, the assessment of lytic activity demonstrated great therapeutic potential (15-47 % coverage). Elvirus, Phikzvirus (jumbophages) were underestimated with DPA (23% coverage) but lysed well in PKA (65% coverage). Many siphoviruses (mainly lysogenic) were overestimated by DPA and lysed only a few strains in PKA. Pbunavirus, Phikzvirus and Bruynoghevirus together showed an improvement in bacterial load applied sequentially and simultaneously in some strains.Summary: Both methods have limitations. We choose PKA as standard method for phage therapy as it is automatable, independent of size/ morphology of the plaque, dependent on lifecycle of phage and comes closest to the situation in the patient. Regarding therapy regime, it is more important which phages are combined than whether it is carried out sequentially or simultaneously.

Combining a phage cocktail with antibiotics improves recovery from Pseudomonas-induced VAP in mice

Ventilator-associated pneumonia (VAP) poses a severe threat in hospital settings, particularly with the rise of multidrug-resistant bacteria like carbapenem-resistant Pseudomonas aeruginosa. Recognized as &quot;critical priority&quot; bacteria by the WHO, these pathogens demand innovative solutions. Our study explores the potential of bacteriophage (phage) therapy as a compelling intervention for VAP. Previously, we showed that even repetitive injections of a P. aeruginosa phage cocktail elicited only a marginal immune response in naïve mice.To assess its therapeutic efficacy, we induced P. aeruginosa-VAP in wild-type mice through high-tidal-volume ventilation, triggering ventilator-induced lung injury, followed by intratracheal infection with P. aeruginosa strain PaO1. Therapy efficacy of the P. aeruginosa-specific phage cocktail, meropenem, or a combination of both via two intraperitoneal administrations was compared.At 24 hpi, the combination therapy effectively reduced the bacterial load up to 3 logs in BAL and lungs, as did meropenem, but showed minimal inflammation and even faster recovery of the mice than under phage cocktail therapy. The clinical score decreased with increasing body temperatures as early as 16 hpi, whereas meropenem-treated mice did not reach normal levels until 24 hpi. Moreover, inflammatory mediators and lung permeability were reduced, but not immune cells in alveolar spaces after phage treatment.In conclusion, our findings propose the synergistic potential of a specific phage cocktail combined with antibiotic treatment as a promising avenue for managing VAP infections caused by P. aeruginosa. This dual approach not only effectively combats the bacterial load, but also contributes to a swifter recovery with reduced inflammation.

Symposium
Fundamentals in Phage Biology and Applications

Appointment

Chair

Topic

Programme

SymposiumFundamentals in Phage Biology and Applications

Appointment

Chair

Topic

Programme

Symposium
Fundamentals in Phage Biology and Applications