Peder Worning

Multiple hospital outbreaks of vanA Enterococcus faecium in Denmark, 2012–13, investigated by WGS, MLST and PFGE

OBJECTIVESnIn Denmark, the incidence of vancomycin-resistant Enterococcus faecium (VREfm) has increased since 2012. The aim of this study was to investigate the epidemiology and clonal relatedness of VREfm isolates in Danish hospitals in 2012-13 using WGS. The second aim was to evaluate if WGS-based typing could replace PFGE for typing of VREfm.nnnMETHODSnA population-based study was conducted including all VREfm isolates submitted for national surveillance from January 2012 to April 2013. All isolates were investigated by WGS, MLST and PFGE.nnnRESULTSnOne-hundred and thirty-two isolates were included. The majority of the isolates were from clinical samples (77%). Gastroenterology/abdominal surgery (29%) and ICUs (29%) were the predominant departments with VREfm. Genomics revealed a polyclonal structure of the VREfm outbreak. Seven subgroups of 3-44 genetically closely related isolates (separated by <17 SNPs) were identified using WGS. Direct or indirect transmission of VREfm between patients and intra- and inter-regional spreading clones was observed. We identified 10 STs. PFGE identified four major clusters (13-43 isolates) and seven minor clusters (two to three isolates). The results from the typing methods were highly concordant. However, WGS-based typing had the highest discriminatory power.nnnCONCLUSIONSnThis study emphasizes the importance of infection control measures to limit transmission of VREfm between patients. However, the diversity of the VREfm isolates points to the fact that other important factors may also affect the VREfm increase in Denmark. Finally, WGS is suitable for typing of VREfm and has replaced PFGE for typing of VREfm in Denmark.

Comparing Whole-Genome Sequencing with Sanger Sequencing for spa Typing of Methicillin-Resistant Staphylococcus aureus

ABSTRACT spa typing of methicillin-resistant Staphylococcus aureus (MRSA) has traditionally been done by PCR amplification and Sanger sequencing of the spa repeat region. At Hvidovre Hospital, Denmark, whole-genome sequencing (WGS) of all MRSA isolates has been performed routinely since January 2013, and an in-house analysis pipeline determines the spa types. Due to national surveillance, all MRSA isolates are sent to Statens Serum Institut, where the spa type is determined by PCR and Sanger sequencing. The purpose of this study was to evaluate the reliability of the spa types obtained by 150-bp paired-end Illumina WGS. MRSA isolates from new MRSA patients in 2013 (n = 699) in the capital region of Denmark were included. We found a 97% agreement between spa types obtained by the two methods. All isolates achieved a spa type by both methods. Nineteen isolates differed in spa types by the two methods, in most cases due to the lack of 24-bp repeats in the whole-genome-sequenced isolates. These related but incorrect spa types should have no consequence in outbreak investigations, since all epidemiologically linked isolates, regardless of spa type, will be included in the single nucleotide polymorphism (SNP) analysis. This will reveal the close relatedness of the spa types. In conclusion, our data show that WGS is a reliable method to determine the spa type of MRSA.

Genomic analysis of 495 vancomycin-resistant Enterococcus faecium reveals broad dissemination of a vanA plasmid in more than 19 clones from Copenhagen, Denmark

Objectives From 2012 to 2014, there has been a huge increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) in Copenhagen, Denmark, with 602 patients infected or colonized with VREfm in 2014 compared with just 22 in 2012. The objective of this study was to describe the genetic epidemiology of VREfm to assess the contribution of clonal spread and horizontal transfer of the vanA transposon (Tn1546) and plasmid in the dissemination of VREfm in hospitals. Methods VREfm from Copenhagen, Denmark (2012–14) were whole-genome sequenced. The clonal structure was determined and the structure of Tn1546-like transposons was characterized. One VREfm isolate belonging to the largest clonal group was sequenced using long-read technology to close a 37 kb vanA plasmid. Results Phylogeny revealed a polyclonal structure where 495 VREfm isolates were divided into 13 main groups and 7 small groups. The majority of the isolates were located in three groups (nu200a=u200a44, 100 and 218) and clonal spread of VREfm between wards and hospitals was identified. Five Tn1546-like transposon types were identified. A dominant truncated transposon (type 4, 92%) was spread across all but one VREfm group. The closed vanA plasmid was highly covered by reads from isolates containing the type 4 transposon. Conclusions This study suggests that it was the dissemination of the type 4 Tn1546-like transposon and plasmid via horizontal transfer to multiple populations of E. faecium, followed by clonal spread of new VREfm clones, that contributed to the increase in and diversity of VREfm in Danish hospitals.

Kinetics of the renneting reaction followed by measurement of turbidity as a function of wavelength

In order to describe the kinetics of rennet coagulation, measurementsn of turbidity as a function of wavelength were used to determine the weight-average degree of polymerization, x¯ w , during renneting of milk at three different concentrations of enzyme and three concentrations of casein, including the normal casein concentration of milk. The change of x¯ w as a function of time was described using Von Smoluchowskis equation, testing a number of expressions for the aggregationn rate constant, k ij . The best description was achieved when k ij was taken as a functionn of an energy barrier against aggregation that was diminished by the proteolysisn of κ-casein. The initial value of the energy barrier partly dependedn on the casein concentration, and had a value >25 k B T at normal casein concentration, where k B is Boltzmanns constant and T the absolute temperature. Whenn the proteolysis of κ-casein was complete, the energy barrier was reduced to 11 k B T and was independent of casein concentration.

Whole Genome Sequencing of Danish Staphylococcus argenteus Reveals a Genetically Diverse Collection with Clear Separation from Staphylococcus aureus

Staphylococcus argenteus (S. argenteus) is a newly identified Staphylococcus species that has been misidentified as Staphylococcus aureus (S. aureus) and is clinically relevant. We identified 25 S. argenteus genomes in our collection of whole genome sequenced S. aureus. These genomes were compared to publicly available genomes and a phylogeny revealed seven clusters corresponding to seven clonal complexes. The genome of S. argenteus was found to be different from the genome of S. aureus and a core genome analysis showed that ~33% of the total gene pool was shared between the two species, at 90% homology level. An assessment of mobile elements shows flow of SCCmec cassettes, plasmids, phages, and pathogenicity islands, between S. argenteus and S. aureus. This dataset emphasizes that S. argenteus and S. aureus are two separate species that share genetic material.

Draft genome sequence of a Kluyvera intermedia isolate from a patient with a pancreatic abscess

The genus Kluyvera comprises potential pathogens that can cause many infections. This study reports a Kluyvera intermedia strain (FOSA7093) from a pancreatic cyst specimen from a long-term hospitalised patient. Whole-genome sequencing (WGS) of the K. intermedia isolate was performed and the strain was reported as sensitive to Danish-registered antibiotics although it had a fosA-like gene in the genome. There were nine contigs that aligned to a plasmid, and these contigs contained several heavy metal resistance gene homologues. Furthermore, a prophage was discovered in the genome. WGS represents an efficient tool for monitoring Kluyvera spp. and its role as a reservoir of multidrug resistance. Therefore, this susceptible K. intermedia genome has many characteristics that allow comparison of resistant K. intermedia that might be discovered in the future.

Complete Genome Sequence of Staphylococcus aureus Strain M1, a Unique t024-ST8-IVa Danish Methicillin-Resistant S. aureus Clone

ABSTRACT We report the genome sequence, in five contigs, of a methicillin-resistant Staphylococcus aureus isolate designated M1. This clinical isolate was from the index patient of a methicillin-resistant Staphylococcus aureus (MRSA) outbreak in Copenhagen, Denmark, that started in 2003. This strain is sequence type 8 (ST8), spa type t024, and staphylococcal cassette chromosome mec element (SCCmec) type IVa.

Typing of vancomycin-resistant enterococci with MALDI-TOF mass spectrometry in a nosocomial outbreak setting

OBJECTIVESnTo investigate the usefulness of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) typing as a first-line epidemiological tool in a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VREfm).nnnMETHODSnFifty-five VREfm isolates, previously characterized by whole-genome sequencing (WGS), were included and analysed by MALDI-TOF MS. To take peak reproducibility into account, ethanol/formic acid extraction and other steps of the protocol were conducted in triplicate. Twenty-seven spectra were generated per isolate, and spectra were visually inspected to determine discriminatory peaks. The presence or absence of these was recorded in a peak scheme.nnnRESULTSnNine discriminatory peaks were identified. A characteristic pattern of these could distinguish between the three major WGS groups: WGS I, WGS II and WGS III. Only one of 38 isolates belonging to WGS I, WGS II or WGS III was misclassified. However, ten of the 17 isolates not belonging to WGS I, II or III displayed peak patterns indistinguishable from those of the outbreak strain.nnnCONCLUSIONSnUsing visual inspection of spectra, MALDI-TOF MS typing proved to be useful in differentiating three VREfm outbreak clones from each other. However, as non-outbreak isolates could not be reliably differentiated from outbreak clones, the practical value of this typing method for VREfm outbreak management was limited in our setting.

Evolution of the β-lactam-resistant Streptococcus pneumoniae PMEN3 clone over a 30 year period in Barcelona, Spain

ObjectivesnTo analyse the epidemiology and genetic evolution of PMEN3 (Spain9V-156), a penicillin-non-susceptible clone of Streptococcus pneumoniae, causing invasive pneumococcal disease (IPD) in Barcelona during 1987-2016.nnnMethodsnWGS was performed on 46 representative isolates and the data were used to design additional molecular typing methods including partial MLST, PCR-RFLP and detection of surface-exposed proteins and prophages, to assign the remaining isolates to lineages. The isolates were also subjected to antimicrobial susceptibility testing.nnnResultsnTwo hundred and twenty-seven adult cases of IPD caused by PMEN3 were identified. PMEN3 caused mainly pneumonia (84%) and the 30u2009day mortality rate was 23.1%. Evidence of recombination events was found, mostly in three regions, namely the capsular operon (associated with capsular switching) and adjacent regions containing pbp2x and pbp1a, the murM gene and the pbp2b-ddl region. Some of these genetic changes generated successful new variant serotype lineages, including one of serotype 11A that is not included in the current PCV13 vaccine. Other genetic changes led to increased MICs of β-lactams. Notably, most isolates also harboured prophages coding for PblB-like proteins. Despite these adaptations, the ability of this clone to cause IPD remained unchanged over time, highlighting the importance of its core genetic background.nnnConclusionsnOur study demonstrated successful adaptation of PMEN3 to persist over time despite the introduction of broader antibiotics and conjugate vaccines. In addition to enhancing understanding of the molecular evolution of PMEN3, these findings highlight the need for the development of non-serotype-based vaccines to fight pneumococcal infection.