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Bacterial viruses targeting multi-resistant Klebsiella pneumoniae and Escherichia coli
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Nilsson)
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The global increase in antibiotic resistance levels in bacteria is a growing concern to our society and highlights the need for alternative strategies to combat bacterial infections. Bacterial viruses (phages) are the natural predators of bacteria and are as diverse as their hosts, but our understanding of them is limited. The current levels of knowledge regarding the role that phage play in the control of bacterial populations are poor, despite the use of phage therapy as a clinical therapy in Eastern Europe.

The aim of this doctoral thesis is to increase knowledge of the diversity and characteristics of bacterial viruses and to assess their potential as therapeutic agents towards multi-resistant bacteria.

Paper I is the product of de novo sequencing of newly isolated phages that infect and kill multi-resistant Klebsiella pneumoniae. Based on similarities in gene arrangement, lysis cassette type and conserved RNA polymerase, the creation of a new phage genus within Autographivirinae is proposed.

Paper II describes the genomic and proteomic analysis of a phage of the rare C3 morphotype, a Podoviridae phage with an elongated head that uses multi-resistant Escherichia coli as its host.

Paper III describes the study of a pre-made phage cocktail against 125 clinical K. pneumoniae isolates. The phage cocktail inhibited the growth of 99 (79 %) of the bacterial isolates tested. This study also demonstrates the need for common methodologies in the scientific community to determine how to assess phages that infect multiple serotypes to avoid false positive results.

Paper IV studies the effects of phage predation on bacterial virulence: phages were first allowed to prey on a clinical K. pneumoniae isolate, followed by the isolation of phage-resistant bacteria. The phage resistant bacteria were then assessed for their growth rate, biofilm production in vitro. The virulence of the phage resistant bacteria was then assessed in Galleria mellonella. In the single phage treatments, two out of four phages showed an increased virulence in the in G. mellonella, which was also linked to an increased growth rate of the phage resistant bacteria. In multi-phage treatments however, three out of five phage cocktails decreased the bacterial virulence in G. mellonella compared to an untreated control.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2015. , 49 p.
Keyword [en]
Bacterial viruses, Bacteriophage, Phage, Phage therapy, multi-resistant bacteria, Klebsiella pneumoniae
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-116711ISBN: 978-91-7649-123-2 (print)OAI: oai:DiVA.org:su-116711DiVA: diva2:807580
Public defence
2015-05-29, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20C, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2015-05-07 Created: 2015-04-23 Last updated: 2015-06-23Bibliographically approved
List of papers
1. A suggested new bacteriophage genus, “Kp34likevirus”, within the Autographivirinae subfamily of Podoviridae
Open this publication in new window or tab >>A suggested new bacteriophage genus, “Kp34likevirus”, within the Autographivirinae subfamily of Podoviridae
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2015 (English)In: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 7, no 4, 1804-1822 p.Article, review/survey (Refereed) Published
Abstract [en]

Klebsiella pneumoniae phages vB_KpnP_SU503 (SU503) and vB_KpnP_SU552A (SU552A) are virulent viruses belonging to theAutographivirinae subfamily of Podoviridae that infect and kill multi-resistant K. pneumoniae isolates. Phages SU503 and SU552A show high pairwise nucleotide identity to Klebsiella phages KP34 (NC_013649), F19 (NC_023567) and NTUH-K2044-K1-1 (NC_025418). Bioinformatic analysis of these phage genomes show high conservation of gene arrangement and gene content, conserved catalytically active residues of their RNA polymerase, a common and specific lysis cassette, and form a joint cluster in phylogenetic analysis of their conserved genes. Also, we have performed biological characterization of the burst size, latent period, host specificity (together with KP34 and NTUH-K2044-K1-1), morphology, and structural genes as well as sensitivity testing to various conditions. Based on the analyses of these phages, the creation of a new phage genus is suggested within the Autographivirinae, called “Kp34likevirus” after their type phage, KP34. This genus should encompass the recently genome sequenced Klebsiella phages KP34, SU503, SU552A, F19 and NTUH-K2044-K1-1.

Keyword
“Kp34likevirus”, phikmvlikevirus, Autographivirinae, Klebsiella pneumoniae, bacteriophage, phage, Podoviridae
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-115896 (URN)10.3390/v7041804 (DOI)000353720400013 ()
Available from: 2015-04-08 Created: 2015-04-08 Last updated: 2017-12-04Bibliographically approved
2. Genomic, Proteomic, Morphological, and Phylogenetic Analyses of vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology
Open this publication in new window or tab >>Genomic, Proteomic, Morphological, and Phylogenetic Analyses of vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 12, e116294Article in journal (Refereed) Published
Abstract [en]

A recently isolated phage, vB_EcoP_SU10 (SU10), with the unusual elongated C3 morphotype, can infect a wide range of Escherichia coli strains. We have sequenced the genome of this phage and characterized it further by mass spectrometry based proteomics, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and ultra-thin section electron microscopy. The genome size is 77,327 base pairs and its genes, and genome architecture, show high similarity to the phiEco32 phage genes and genome. The TEM images reveal that SU10 have a quite long tail for being a Podoviridae phage, and that the tail also changes conformation upon infection. The ultra-thin section electron microscopy images of phages at the stage of replication within the host cell show that the phages form a honeycomb-like structure under packaging of genomes and assembly of mature capsids. This implies a tight link between the replication and cutting of the concatemeric genome, genome packaging, and capsid assembly. We have also performed a phylogenetic analysis of the structural genes common between Podoviridae phages of the C1 and C3 morphotypes. The result shows that the structural genes have coevolved, and that they form two distinct groups linked to their morphotypes. The structural genes of C1 and C3 phages appear to have diverged around 280 million years ago applying a molecular clock calibrated according to the presumed split between the Escherichia - Salmonella genera.

National Category
Biological Sciences Microbiology in the medical area
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-113563 (URN)10.1371/journal.pone.0116294 (DOI)000347119100128 ()
Note

AuthorCount:5;

Available from: 2015-02-05 Created: 2015-02-04 Last updated: 2017-12-05Bibliographically approved
3. A novel phage cocktail inhibiting the growth of 99 β-lactamase carrying Klebsiella pneumoniae clinical isolates in vitro
Open this publication in new window or tab >>A novel phage cocktail inhibiting the growth of 99 β-lactamase carrying Klebsiella pneumoniae clinical isolates in vitro
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Klebsiella pneumoniae is a gram-negative bacterial pathogen, accountable for a variety of nosocomial infections in immunocompromised patients, open-wound infections and community-acquired pneumonia in elderly. K. pneumoniae strains harboring plasmid-mediated extended spectrum β-lactamase enzymes (ESBL) are resistant to all penicillin and cephalosoprins, whereas bacteria capable of producing carbapenemase enzymes (e.g. NDM, KPC and VIM) are resistant to virtually all β-lactam group antibiotics. The use of bacterial viruses lysing bacterial hosts (phage therapy) has been suggested as an alternative in fighting bacterial infections resistant to known antibiotics. In this study, we assembled a phage cocktail consisting of 6 novel lytic bacteriophages infecting K. pneumoniae. The phage cocktail was tested against 125 β-lactamase producing clinical isolates of K. pneumoniae and we found that at high titres, the cocktail was able to lyse 99 of these isolates in vitro.

Keyword
Phage therapy, Klebsiella pneumoniae, Lytic phage, ESBL, Phage cocktail
National Category
Microbiology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-115898 (URN)
Available from: 2015-04-08 Created: 2015-04-08 Last updated: 2016-01-29Bibliographically approved
4. Evolution of virulence in Klebsiella pneumoniae treated with phage cocktails
Open this publication in new window or tab >>Evolution of virulence in Klebsiella pneumoniae treated with phage cocktails
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The worldwide emergence and spread of multidrug-resistant bacteria is a major concern in modern medicine, and threatens the once established control over bacterial infections. Phage therapy has been suggested as one potential solution to the problem of finding new antibacterial agents. Bacteria are known to evolve resistance against bacteriophages but in many cases phage-resistance comes with a cost on bacterial virulence in multicellular hosts. We investigated how the virulence of a clinical isolate of K. pneumoniae Kpn524 evolves in response to exposure to phage cocktails in the phage-resistant bacteria that would potentially survive the phage treatment. We found that the exposure to multiple phages was linked to lowered virulence in the phage-resistant bacteria, when measured in vivo with Galleria mellonella. However, two phages were found to increase the bacterial virulence when they were administered on the bacteria individually, and this was associated with an increased growth rate. Across all treatments, biofilm production was negatively correlated with virulence, whereas growth rate had a positive correlation with bacterial virulence. Our findings suggest that bacterial virulence is attenuated in the presence of multiple phages, possibly due to a trade-off between phage resistance andrate of replication. However, this is dependent on the composition of the phage cocktail.This is the first study to report increased bacterial virulence associated with exposure tolytic bacteriophages and our results call for meticulous consideration when choosingphages for phage cocktails, as phages with certain identity could have detrimentally adverse effects on the success of the treatment.

National Category
Evolutionary Biology Genetics Microbiology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-115902 (URN)
Available from: 2015-04-08 Created: 2015-04-08 Last updated: 2016-01-29Bibliographically approved

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