Christian Giske group

Exploring why some clones are successful

Certain clones of extensively drug resistant (XDR) Klebsiella pneumoniae are frequently linked to the dissemination of resistance genes, as well as causing outbreaks. Such clones have been defined by multi-locus sequence typing (MLST) to belong to e.g to sequence types (ST) 258, 14, and 147. However, little is still known about why these clones are successful. In this project, we are exploring two explanations; prophages, bacteriophages integrated in the genome, and the presence of CRISPR/Cas, a “bacterial immune system”.

Bacterial clones are subjected to whole-genome sequencing and the sequence data is used to look for differences explaining the success of specific clones. This is followed by in vivo experiments with waxmoth larvae (Galleria mellonella) and lastly, competition experiments followed by analysis with fluorescence-activated cell sorter (FACS). XDR K. pneumoniae strains are also used to spike feces samples to investigate the potential of curing feces of such strains.

Understanding the interaction between successful clones and the microbiota

The intestinal microbiome serves as a reservoir for carriage of antimicrobial resistant bacteria, such as extended-spectrum β-lactamase (ESBL) producing Escherichia coli (EP-EC). Specific phylogenetic clonal linages in E. coli, such as phylogroup B2, sequence type (ST) 131 and the ST131 subclone H30-Rx have been linked to pandemic spread and increased potential to cause severe infections because of higher virulence.

In this project, we investigate if successful clonal lineages in the fecal microbiota are prone to develop a long-term carriage and increase the risk disease, and if this has implications on the diversity of the fecal microbiota. EP-EC strains isolated from patients are subjected to whole-genome sequencing and this is followed by metagenomic characterisation to evaluate species diversity. In vitro competition assays are used to experimentally demonstrate whether some clones can outcompete other commensal clones. The results are used to facilitate risk stratification of fecal carriage and identify interventions against high-risk carriage, such bacteriophage treatment of E. coli in the microbiota.

Importance of antimicrobial stewardship for decreasing the risk of dominance of the fecal microbiota by colonising resistant strains

In this project, we assess the impact of prescription quality, infection control, and antimicrobial stewardship on gut microbiota domination by healthcare-associated pathogens. This is a comprehensive, multinational, multi-centre clinical study aiming to assess the impact of inadequate antibiotic prescription on intestinal domination by extended-spectrum beta-lactamase producing Enterobacteriaceae, vancomycin-resistant enterococci, or infection with C. difficile. The study follows the progression from first acquisition of drug-resistant organisms to infection with these bacteria at individual patient level. Changes in the microbiota are assessed both with 16S rDNA metagenomics sequencing, and with phenotypic microbiota characterisation. This study is strictly observational, meaning that it has no influence on the selection of antimicrobial treatment. Patient stool samples are collected up to a maximum of seven times for included patients developing fever or receiving antibiotic treatment. All patients with a high likelihood of needing an antibiotic in the near future are eligible for the study. To avoid biases, patients who may already have a disturbed gut microbiota, e.g. due to recent antibiotic use or diarrheal conditions, are not included in the study.

Tracking plasmids directly from clinical specimens

The goal of this project is to establish a recently developed method for plasmids characterisation to clinical diagnostics. The method is based on novel nanotechnology and can therefore work with very small quantities of DNA, which makes it possible to analyse samples with only a short pre-cultivation step. We work directly on clinical samples from ICU-patients and conduct a comparison between the novel technique and a culture-based approach. The goal of this project is also to incorporate the technology into a low- cost device based on a smart phone microscope, which will allow for a broader use of the technique and also transfer to low-income settings.

Therapeutic antibodies against extensively drug-resistant (XDR) K.pneumoniae

Therapeutic antibodies have proven an effective method to treat Clostridium difficile and methicillin-resistant Staphylococcus aureus (MRSA). In this project, we explore the potential role of monoclonal antibodies (mAb) for counteracting XDR K. pneumoniae.

The project is aimed at developing a specific efficacy assay for various strains of K. pneumoniae. The mAb is tested on isolates of various types of resistant K. pneumoniae. The antibody’s efficacy is studied in an in vitro lung cell model. Also, mechanism of action studies are performed on the lead antibodies with regards to K. pneumoniae activity. Possible non-respondent strains are analysed and possible resistance mechanisms studied by genome sequencing, mapping also chromosomal mechanisms.

Subsequently mAbs will are tested in an immunocompromised mouse model for their protective, effect following challenge with K. pneumoniae strains selected from the collection of the research group.

Antimicrobial susceptibility testing with calorimetrics

In this project, we use a calorimetric device (calScreener, Symcel Sverige Ab) to detect bacterial metabolism in the presence of antimicrobials for the purpose of antimicrobial susceptibility testing.

The project consists of three parts:

1. Validation of calScreener on stored patient isolates (broth microdilution) with known antimicrobial susceptibility, focusing on gram-negative bacilli and the most clinically used antimicrobials.
2. Retrospective study, focusing susceptibility testing of extensively drug-resistant gram-negative bacilli from stored patient isolates, using a combination of antibiotics (synergy testing) as well as single agents. To evaluate the accuracy of the calorimetric assay, results will be compared with animal models
3. Prospective observational study to investigate whether calorimetry can be used as a standard method in a clinical environment for antimicrobial susceptibility testing using clinical isolates from patients with confirmed bloodstream infection.

This method is also used in the development antimicrobial susceptibility testing for bacteriophages and antimicrobial peptides.

Projects Göran Kronvall

Read more about the Göran Kronvall projects

Group members

Affiliated

Associated

Sara Byfors
Erja Chryssanthou
Petra Edquist
Hong Fang
Wilhelm Hedin
Karolina Hedman
Mirja Hägg
Aina Iversen
Göran Kronvall
Öjar Melefors
Mamun Rashid

Funding

• Horizon 2020 (BADGER)
• Eurostars/Vinnova (SPARKLE)
• Joint Programme Initiative for Antimicrobial Resistance/Swedish Research Council (PILGRIM)
• Swedish Research Council (project funding and project in Southeast Asia)
• Swedish International Development Cooperation Agency
• Swedish Medical Society
• Stockholm County
• Erling Persson Foundation

Publications

Selected publications

Invasive infection caused by Klebsiella pneumoniae is a disease affecting patients with high comorbidity and associated with high long-term mortality.
Vading M, Nauclér P, Kalin M, Giske CG
PLoS ONE 2018 ;13(4):e0195258

Community carriage of ESBL-producing Escherichia coli is associated with strains of low pathogenicity: a Swedish nationwide study.
Ny S, Löfmark S, Börjesson S, Englund S, Ringman M, Bergström J, et al
J. Antimicrob. Chemother. 2017 02;72(2):582-588

Frequent acquisition of low-virulence strains of ESBL-producing Escherichia coli in travellers.
Vading M, Kabir MH, Kalin M, Iversen A, Wiklund S, Nauclér P, et al
J. Antimicrob. Chemother. 2016 12;71(12):3548-3555

Metagenomic analysis of bloodstream infections in patients with acute leukemia and therapy-induced neutropenia.
Gyarmati P, Kjellander C, Aust C, Song Y, Öhrmalm L, Giske CG
Sci Rep 2016 Mar;6():23532

Carbapenemase-producing Enterobacteriaceae in Sweden 2007-2013: Experiences from seven years of systematic surveillance and mandatory reporting.
Löfmark S, Sjöström K, Mäkitalo B, Edquist P, Tegmark Wisell K, Giske CG
Drug Resist. Updat. 2015 May;20():29-38

Klebsiella variicola is a frequent cause of bloodstream infection in the stockholm area, and associated with higher mortality compared to K. pneumoniae.
Maatallah M, Vading M, Kabir MH, Bakhrouf A, Kalin M, Nauclér P, et al
PLoS ONE 2014 ;9(11):e113539

Faecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae is common 12 months after infection and is related to strain factors.
Titelman E, Hasan CM, Iversen A, Nauclér P, Kais M, Kalin M, et al
Clin. Microbiol. Infect. 2014 Aug;20(8):O508-15

Epidemiology of extended-spectrum β-lactamase-producing Escherichia coli in Sweden 2007-2011.
Brolund A, Edquist PJ, Mäkitalo B, Olsson-Liljequist B, Söderblom T, Wisell KT, et al
Clin. Microbiol. Infect. 2014 Jun;20(6):O344-52

Dissemination of blaVIM in Greece at the peak of the epidemic of 2005-2006: clonal expansion of Klebsiella pneumoniae clonal complex 147.
Hasan CM, Turlej-Rogacka A, Vatopoulos AC, Giakkoupi P, Maâtallah M, Giske CG
Clin. Microbiol. Infect. 2014 Jan;20(1):34-7

Diverse sequence types of Klebsiella pneumoniae contribute to the dissemination of blaNDM-1 in India, Sweden, and the United Kingdom.
Giske CG, Fröding I, Hasan CM, Turlej-Rogacka A, Toleman M, Livermore D, et al
Antimicrob. Agents Chemother. 2012 May;56(5):2735-8

Christian G. Giske’s publications on PubMed

https://www.ncbi.nlm.nih.gov/pubmed/?term=Giske%20CG%5BAuthor%5D&cauthor=true&cauthor_uid=29518198

Visiting address

Free downloads

Single strain regression analysis programme for calibration of disk diffusion testing, etc (3.9 Mb installation file).