Mobile genetic elements causing plasticity in E. faecium

The paper 3 of this thesis is not available in Munin. Paper 3: Sivertsen, A., Pedersen, T., Janice, J., Hegstad, K.: “The Enterococcus Cassette Chromosome: an SCCmec-like mobilisable element”. (Manuscript).I helseinstitusjoner vancomycin-resistente enterokokker en fryktet bakterie som kan lage alvorlig infeksjonssykdom i pasienter med dårlig immunforsvar, og er vanskelig å behandle. Avhandlingen fokuserer på vancomycin-resistente enterokokker (VRE), og hvordan disse bakteriene gjennom å overføre gener mellom hverandre kan utvikle resistens mot antibiotika. Vi har analysert to utbrudd av VRE i Sverige og i Norge, og har funnet at VRE kan være på vei til å bli et mer vanlig patogen i skandinaviske sykehus. Dagens diagnostiske verktøy er ikke i stand til å fange opp alle typer VRE. Dette har konsekvenser for både diagnostikk, resistensovervåkning og risiko for feilbehandling av svært syke pasienter. Nye gensekvenseringsteknologier kan forbedre denne type diagnostikk ved å kunne se på genotypen i tillegg, noe som er viktig da vancomycinresistensgener kan være tilstede i bakterien uten at bakterien har fenotype for dette. I begge utbruddene var diagnostikken utilfredsstillende, da bakterien kunne bli tolket som følsom. I det ene utbruddet utviklet VRE resistens under behandling på grunn av for eksempel mobile genetiske elementer kalt IS-elementer hoppet rundt i vancomycinresistensgenene og påvirket uttrykket deres. Resultater fra doktorgraden har allerede ført til en endring i rådgiving på diagnostikk av VRE hos svært syke pasienter. Vi har også funnet at mobile genetiske elementer (MGE), DNA-et som flytter seg mellom bakterier, har en stor evne til å rekombinere seg og danne varianter med ulikt geninnhold. Inntil nå har strukturen på disse elementene vært vanskelig å rekonstruere. Dermed har betydningen av MGE’er for bakterienes resistenspotensiale vært vanskelig å vurdere. Man kan observere at disse elementene oppfører seg som Babushka-dukker ved å koble seg på hverandre. Såkalte long-read sekvenseringsmetoder er i stand til å rekonstruere strukturen på slike elementer, og det at de kun har vært kommersielt tilgjengelige de siste 3-4 år gjør at der fortsatt er mye å lære om mobile genetiske elementer og deres innvirkning på resistensutvikingen av bakterier

Genomic epidemiology of Streptococcus dysgalactiae subsp. equisimilis strains causing invasive disease in Norway during 2018

Background: Streptococcus dysgalactiae subspecies equisimilis (SDSE) is an emerging global pathogen, yet the epidemiology and population genetics of SDSE species have not been extensively characterized. Methods: We carried out whole genome sequencing to characterize 274 SDSE isolates causing bloodstream infections obtained through national surveillance program in 2018. We conducted multilocus sequence typing (MLST), emm-typing, core genome phylogeny, as well as investigated key features associated with virulence. Moreover, comparison to SDSE from other geographic regions were performed in order to gain more insight in the evolutionary dynamics in SDSE. Results: The phylogenetic analysis indicated a substantial diversity of emm-types and sequence types (STs). Briefly, 17 emm-types and 58 STs were identified that formed 10 clonal complexes (CCs). The predominant ST-types were ST20 (20%), ST17 (17%), and ST29 (11%). While CC17 and CC29 clades showed a substantial heterogeneity with well-separated emm-associated subclades, the CC20 clade harboring the stG62647 emm-type was more homogenous and the most prevalent in the present study. Moreover, we observed notable differences in the distribution of clades within Norway, as well as several disseminated CCs and also distinct geographic variations when compared to data from other countries. We also revealed extensive intra-species recombination events involving surface exposed virulence factors, including the emm gene important for phylogenetic profiling. Conclusion: Recombination events involving the emm as well as other virulence genes in SDSE, are important mechanisms in shaping the genetic variability in the SDSE population, potentially offering selective advantages to certain lineages. The enhanced phylogenetic resolution offered by whole genome sequencing is necessary to identify and delimitate outbreaks, monitor and properly characterize emerging strains, as well as elucidate bacterial population dynamics.publishedVersio

Effect of leukoreduction and temperature on risk of bacterial growth in CPDA-1 whole blood: A study of Escherichia coli

Background Collection of non-leukoreduced citrate-phosphate-dextrose-adenine (CPDA-1) whole blood is performed in walking blood banks. Blood collected under field conditions may have increased risk of bacterial contamination. This study was conducted to examine the effects of WBC reduction and storage temperature on growth of Escherichia coli (ATCC® 25922™) in CPDA-1 whole blood. Methods CPDA-1 whole blood of 450 ml from 10 group O donors was inoculated with E. coli. Two hours after inoculation, the test bags were leukoreduced with a platelet-sparing filter. The control bags remained unfiltered. Each whole blood bag was then split into three smaller bags for further storage at 2–6°C, 20–24°C, or 33–37°C. Bacterial growth was quantified immediately, 2 and 3 h after inoculation, on days 1, 3, 7, and 14 for all storage temperatures, and on days 21 and 35 for storage at 2–6°C. Results Whole blood was inoculated with a median of 19.5 (range 12.0–32.0) colony-forming units per ml (CFU/ml) E. coli. After leukoreduction, a median of 3.3 CFU/ml (range 0.0–33.3) E. coli remained. In the control arm, the WBCs phagocytized E. coli within 24 h at 20–24°C and 33–37°C in 9 of 10 bags. During storage at 2–6°C, a slow self-sterilization occurred over time with and without leukoreduction. Conclusions Storage at 20–24°C and 33–37°C for up to 24 h before leukoreduction reduces the risk of E. coli-contamination in CPDA-1 whole blood. Subsequent storage at 2–6°C will further reduce the growth of E. coli.publishedVersio

Possible origin of a vanB-type Enterococcus faecium causing a multicentre outbreak in Sweden

Dissemination of vancomycin-resistant Enterococcus faecium (VREfm) with similar pulsed-field gel electrophoresis patterns in three Swedish hospitals between 2007 and 2011 prompted molecular characterization to reveal the possible origins and features of the strain. A representative subset of collected isolates (VREfm (n=18) and vancomycin-susceptible Enterococcus faecium (VSEfm) (n=2)) reflecting the spread in time and location was subjected to Multi Locus Sequence Typing, antibiotic resistance testing, virulence gene screening, characterization of mobile genetic units carrying the resistance gene and their ability to transfer. In addition, 3 outbreak strains and 1 isolate collected prior to the outbreak was whole-genome sequenced. The isolates were predominantly ST192, considered to belong within a high-risk lineage, and concordantly harbored at least eight virulence genes associated with high-risk genotypes, as well as were geno- and phenotypically resistant to ampicillin, gentamicin, ciprofloxacin and vancomycin, with susceptibility to teicoplanin. The vancomycin resistance was of vanB2-type, and this gene cluster was part of the conjugative transposon Tn1549/Tn5382. PFGE analysis with S1 nuclease restriction as well as filter mating experiments indicated that vanB2-Tn1549/Tn5382 was placed in a 70 kb sized pRUM replicon, which readily transferred between E. faecium. The plasmid also contained an axe-txe toxin-antitoxin stability module capable of securing persistence within the bacterial host. The two VSEfm were similar by PFGE and MLST and harbored a 30 kb smaller pRUM plasmid lacking the vanB2-Tn1549/Tn5382. In conclusion, the obtained results indicate introduction of vanB2-Tn1549/Tn5382 into a pRUM plasmid harbored in a pre-existing high-risk clone. Afterwards, the resulting VRE containing the pRUM-vanB2-Tn1549/Tn5382-axe-txe plasmid successfully disseminated in the three hospitals

Problemløsningsmønstre. En kvalitativ studie av elevers problemløsningsstrategier for numeriske tallrekker og geometriske mønstre

Denne undersøkelsen har tittelen: Problemløsningsmønstre. Hensikten med oppgaven er å bedre forståelsen av hvilke problemløsningsstrategier som kommer til syne når elever løser oppgaver med tallrekker innenfor algebrafeltet. Studien tar utgangspunkt i et konstruktivistisk kunnskapssyn, og har et kvalitativt forskningsdesign. Datainnsamlingen fant sted i to 9. klasser ved to skoler. Jeg har tatt i bruk oppgavebasert intervju som metode, og filmet de 10 elevene mens de har løst de ulike oppgavene. Videre transkriberte og kodet jeg datamaterialet, og ut i fra mitt konseptuelle rammeverk har jeg tolket det og sett etter mønster i valg av strategier. Jeg har presentert de ulike strategiene som kommer frem av elevenes løsninger. Gjennom analysen fant jeg at elevene har ganske like strategivalg, for de to første oppgavene, mens de velger flere ulike måter å løse den tredje oppgaven. Av strategivalg for oppgavene er det se etter mønster som elevene benytter seg flest ganger av, som nok henger mye med oppgavenes tema: numeriske rekker og geometriske mønstre. Hvorfor elevene velger disse strategiene er interessant og en kan avdekke elevers syn på matematikkfaget i disse

Pain and pain tolerance in whiplash-associated disorders: A population-based study

Background Pain is a cardinal symptom in individuals with whiplash-associated disorders (WAD). We aimed to compare pain characteristics between individuals with WAD and individuals reporting chronic pain from other causes, and to determine whether potential differences were accounted for by experimental pain tolerance. Methods Data from the 6th Tromsø Study (2007–2008, n = 12,981) were analysed. The number of painful locations was compared between individuals with WAD and individuals reporting chronic pain from other causes using negative binomial regression, pain frequency using multinomial logistic regression and pain intensity using multiple linear regression. Differences in experimental pain tolerance (cold pressor test) were tested using Cox regression; one model compared individuals with WAD to those with chronic pain from other causes, one compared the two groups with chronic pain to individuals without chronic pain. Subsequently, regression models investigating clinical pain characteristics were adjusted for pain tolerance. Results Of individuals with WAD, 96% also reported other causes for pain. Individuals with WAD reported a higher number of painful locations [median (inter-quartile range): 5 (3.5–7) vs. 3 (2–5), p < 0.001] and higher pain intensity (crude mean difference = 0.78, p < 0.001) than individuals with chronic pain from other causes. Pain tolerance did not differ between these two groups. Compared to individuals without chronic pain, individuals with WAD and individuals with chronic pain from other causes had reduced pain tolerance. Conclusions Individuals with WAD report more additional causes of pain, more painful locations and higher pain intensity than individuals with chronic pain from other causes. The increased pain reporting was not accounted for by pain tolerance

Alternative vanHAX promoters and increased vanA-plasmid copy number resurrect silenced glycopeptide resistance in Enterococcus faecium

Background - Vancomycin variable enterococci (VVE) are van-positive isolates with a susceptible phenotype that can convert to a resistant phenotype during vancomycin selection. Objectives - To describe a vancomycin-susceptible vanA-PCR positive ST203 VVE Enterococcus faecium isolate (VVESwe-S) from a liver transplantation patient in Sweden which reverted to resistant (VVESwe-R) during in vitro vancomycin exposure. Methods - WGS analysis revealed the genetic differences between the isolates. Expression of the van-operon was investigated by qPCR. Fitness and stability of the revertant were investigated by growth measurements, competition and serial transfer. Results - The VVESwe-R isolate gained high-level vancomycin (MIC >256 mg/L) and teicoplanin resistance (MIC = 8 mg/L). VVESwe-S has a 5′-truncated vanR activator sequence and the VVESwe-R has in addition acquired a 44 bp deletion upstream of vanHAX in a region containing alternative putative constitutive promoters. In VVESwe-R the vanHAX-operon is constitutively expressed at a level comparable to the non-induced prototype E. faecium BM4147 strain. The vanHAX operon of VVESwe is located on an Inc18-like plasmid, which has a 3–4-fold higher copy number in VVESwe-R compared with VVESwe-S. Resistance has a low fitness cost and the vancomycin MIC of VVESwe-R decreased during in vitro serial culture without selection. The reduction in MIC was associated with a decreased vanA-plasmid copy number. Conclusions - Our data support a mechanism by which vancomycin-susceptible VVE strains may revert to a resistant phenotype through the use of an alternative, constitutive, vanR-activator-independent promoter and a vanA-plasmid copy number increase

Role of Horizontal Gene Transfer in the Development of Multidrug Resistance in Haemophilus influenzae

Haemophilus influenzae colonizes the respiratory tract in humans and causes both invasive and noninvasive infections. Resistance to extended-spectrum cephalosporins in H. influenzae is rare in Europe. In this study, we defined acquired resistance gene loci and ftsI mutations in multidrug-resistant (MDR) and/or PBP3-mediated beta-lactam-resistant (rPBP3) H. influenzae strains, intending to understand the mode of spread of antibiotic resistance determinants in this species. Horizontal transfer of mobile genetic elements and transformation with resistance-conferring ftsI alleles were contributory. We found one small plasmid and three novel integrative conjugative elements (ICEs) which carry different combinations of resistance genes. Demonstration of transfer and/or ICE circular forms showed that the ICEs are functional. Two extensively MDR genetically unrelated H. influenzae strains (F and G) from the same geographical region shared an identical novel MDR ICE (Tn6686) harboring blaTEM-1, catA2-like, and tet(B). The first Nordic case of MDR H. influenzae septicemia, strain 0, originating from the same geographical area as these strains, had a similar resistance pattern but contained another ICE [Tn6687 with blaTEM-1, catP and tet(B)] with an overall structure quite similar to that of Tn6686. Comparison of the complete ftsI genes among rPBP3 strains revealed that the entire gene or certain regions of it are identical in genetically unrelated strains, indicating horizontal gene transfer. Our findings illustrate that H. influenzae is capable of acquiring resistance against a wide range of commonly used antibiotics through horizontal gene transfer, in terms of conjugative transfer of ICEs and transformation of chromosomal genes

Streptococcus dysgalactiae Bloodstream Infections, Norway, 1999–2021

Streptococcus dysgalactiae increasingly is recognized as a pathogen of concern for human health. However, longitudinal surveillance data describing temporal trends of S. dysgalactiae are scarce. We retrospectively identified all β-hemolytic streptococcal bloodstream infections reported in Bergen, in western Norway, during 1999–2021. To explore S. dysgalactiae disease burden in a broader context, we mapped the incidence of all microbial species causing bloodstream infections during 2012–2021. We found S. dysgalactiae incidence rates substantially increased during the study period; by 2021, S. dysgalactiae was the fifth most common pathogen causing bloodstream infections in our region. We noted genotypic shifts and found that the rising trend was related in part to the introduction and expansion of the stG62647 emm-type. S. dysgalactiae is among the most common causes of bloodstream infections in western Norway, and increased surveillance and unambiguous species identification are needed to monitor the disease burden attributable to this pathogen