En studie av mekanismer involvert i Enterococcus faecalis patogenisitet ved hjelp av DNA mikromatriser

Traditionally considered a harmless commensal of the mammalian gastrointestinal tract, Enterococcus faecalis now rank among the leading causes of nosocomial infections worldwide. Several genetic determinants enhancing the virulence of E. faecalis have been characterized, but none of these appears to be indispensible for the pathogenicity of the organism. In this context, the focus of this thesis has been to acquire in-depth knowledge about the specific traits contributing to the success of this versatile pathogen, with DNA microarrays as the main working tool. Microarray-based comparison of gene content in E. faecalis isolated from Norwegian infants, contributed new insight into the genomic repertoire of commensal E. faecalis. Moreover, comparative genomic analysis of a larger collection of isolates identified a set of lineage-enriched genes which may contribute to the fitness of hospital-associated high-risk E. faecalis clonal complex 6 (CC6) strains. Preponderance of phage-related genes among the CC6-enriched genes, suggested a role of genome flexibility in hospital adaptation. Furthermore, significant enrichment of genes encoding surface-related structures may lead to antigenic variation, and may thus supply CC6 strains with a means to evade certain mechanisms of host defense. E. faecalis is intrinsically robust and has a unique ability to adapt to changing environments. Since the pathogenic potential of the bacterium may not only relay on the presence and absence of specific genetic traits, but also on gene regulation, transcriptional profiles were obtained for E. faecalis in the presence of bile, NaCl-induced osmotic shock and growth in urine. In general, these studied highlighted the importance of rapid adaptations in the expression of genes related to energy metabolism, transport systems, cell envelope rearrangements and general stress mechanisms in response to host-relevant growth environments. Moreover, the transcriptional analyses provided clues to hitherto undiscovered mechanisms of resistance associated with the various growth conditions investigated.Enterococcus faecalis, som tradisjonelt sett har vært ansett som en harmløs del av tarmfloraen hos pattedyr, verserer nå på listen over de vanligste årsakene til sykehusinfeksjoner verden over. En rekke genetiske trekk som bidrar til alvorligheten av en E. faecalis infeksjon har blitt karakterisker, men ingen av disse har vist seg å være nødvendige for organismens evne til å forårsake sykdom. På bakgrunn av dette, har fokus for arbeidet omtalt i denne avhandlingen vært å tilegne seg en bedre forståelse av faktorene som bidrar til suksessen til denne allsidige patogenen, men DNA mikromatriser som hovedverktøy. Mikromatrise-baserte sammenligning av geninnhold i E. faecalis isolert fra norske spedbarn, ga økt innsikt i det genetiske repertoaret hos kommensale E. faecalis. Videre, ble det ved hjelp av komparative genomanalyser av en større gruppe isolater identifisert et sett med gener som var anriket blant stammer tilhørende høyrisiko-gruppen klonalt kompleks 6 (CC6). Stammer tilhørende denne gruppen er svært utbredt som årsak til infeksjoner, og da en stor andel av de anrikede genene var lokalisert innefor fag-relaterte områder på kromosomet, kan det tyde på at genomfleksibilitet spiller en viktig rolle i tilpasningen til sykehusmiljøet. Videre utgjorde også gener som koder for overflatestrukturer en signifikant andel av de anrikede genene. Variasjon i disse strukturene kan gi variasjon i antigenisitet, og dermed bidra til at bakterien unnslipper enkelte av vertens forsvarsmekanismer. E. faecalis er en hardfør organisme, og i tillegg svært tilpasningsdyktig. Differensiell regulering av ulike gener kan, på samme måte som forskjeller i geninnhold, bidra til bakteriens patogene potensiale. Transkripsjonsprofiler av E. faecalis dyrket i nærvær at galle, under NaCl-indusert osmotisk stress og i urin understreket viktigheten av tilpasninger i genekspresjon innenfor de funksjonelle kategoriene energimetabolisme, transport, cellevegg/-membran, samt generelle stressmekansimer under disse vertsrelevante vekstforholdene. Videre, ga analysene et innblikk inn i hittil ukjente resistensmekanismer assosiert med de spesifikke vekstbetingelsene.European Union Sixth Framework: LSHE-CT-2007-037410

Comparative genomics of Enterococcus faecalis from healthy Norwegian infants

<p>Abstract</p> <p>Background</p> <p><it>Enterococcus faecalis</it>, traditionally considered a harmless commensal of the intestinal tract, is now ranked among the leading causes of nosocomial infections. In an attempt to gain insight into the genetic make-up of commensal <it>E. faecalis</it>, we have studied genomic variation in a collection of community-derived <it>E. faecalis </it>isolated from the feces of Norwegian infants.</p> <p>Results</p> <p>The <it>E. faecalis </it>isolates were first sequence typed by multilocus sequence typing (MLST) and characterized with respect to antibiotic resistance and properties associated with virulence. A subset of the isolates was compared to the vancomycin resistant strain <it>E. faecalis </it>V583 (V583) by whole genome microarray comparison (comparative genomic hybridization (CGH)). Several of the putative enterococcal virulence factors were found to be highly prevalent among the commensal baby isolates. The genomic variation as observed by CGH was less between isolates displaying the same MLST sequence type than between isolates belonging to different evolutionary lineages.</p> <p>Conclusion</p> <p>The variations in gene content observed among the investigated commensal <it>E. faecalis </it>is comparable to the genetic variation previously reported among strains of various origins thought to be representative of the major <it>E. faecalis </it>lineages. Previous MLST analysis of <it>E. faecalis </it>have identified so-called high-risk enterococcal clonal complexes (HiRECC), defined as genetically distinct subpopulations, epidemiologically associated with enterococcal infections. The observed correlation between CGH and MLST presented here, may offer a method for the identification of lineage-specific genes, and may therefore add clues on how to distinguish pathogenic from commensal <it>E. faecalis</it>. In this work, information on the core genome of <it>E. faecalis </it>is also substantially extended.</p

Enhancement of cranial nerves in Lyme neuroborreliosis: incidence and correlation with clinical symptoms and prognosis

Purpose Symptoms of cranial neuritis are a common presentation of Lyme neuroborreliosis (LNB). Imaging studies are scarce and report contradictory low prevalence of enhancement compared to clinical studies of cranial neuropathy. We hypothesized that MRI enhancement of cranial nerves in LNB is underreported, and aimed to assess the prevalence and clinical impact of cranial nerve enhancement in early LNB. Methods In this prospective, longitudinal cohort study, 69 patients with acute LNB were examined with MRI of the brain. Enhancement of cranial nerves III–XII was rated. MRI enhancement was correlated to clinical findings of neuropathy in the acute phase and after 6 months. Results Thirty-nine of 69 patients (57%) had pathological cranial nerve enhancement. Facial and oculomotor nerves were most frequently affected. There was a strong correlation between enhancement in the distal internal auditory canal and parotid segments of the facial nerve and degree of facial palsy (gamma = 0.95, p < .01, and gamma = 0.93, p < .01), despite that 19/37 nerves with mild-moderate enhancement in the distal internal auditory canal segment showed no clinically evident palsy. Oculomotor and abducens nerve enhancement did not correlate with eye movement palsy (gamma = 1.00 and 0.97, p = .31 for both). Sixteen of 17 patients with oculomotor and/or abducens nerve enhancement had no evident eye movement palsy. Conclusions MRI cranial nerve enhancement is common in LNB patients, but it can be clinically occult. Facial and oculomotor nerves are most often affected. Enhancement of the facial nerve distal internal auditory canal and parotid segments correlate with degree of facial palsy.publishedVersio

Six versus 2 weeks treatment with doxycycline in European Lyme neuroborreliosis: a multicentre, noninferiority, double-blinded, randomised and placebocontrolled trial

Background There is limited evidence regarding optimal duration of antibiotic treatment in neuroborreliosis. We aimed to compare efficacy and safety of oral doxycycline for 2 and 6 weeks in European Lyme neuroborreliosis (LNB). Methods The trial had a randomised, double-blinded, placebo-controlled, non-inferiority design. Patients with LNB were recruited from eight Norwegian hospitals and randomised to doxycycline 200 mg once daily for 2 weeks, followed by 4 weeks of placebo, or doxycycline 200 mg once daily for 6 weeks. The primary endpoint was clinical improvement as measured by difference in a Composite Clinical Score (0–64 points) from baseline to 6 months. The non-inferiority margin was predetermined to 0.5 points. Results One hundred and twenty-one patients were included. Fifty-two treated for 2 weeks and 53 for 6 weeks were included in the intention-to-treat analyses, and 52 and 51 in per-protocol analysis. Mean difference in clinical improvement between the groups was 0.06, 95% CI −1.2 to 1.2, p=0.99 in the intention-to-treat population, and −0.4, 95% CI −1.4 to 0.7, p=0.51 in the per-protocol population and non-inferiority could not be established. There were no treatment failures and no serious adverse events. The groups did not differ in secondary outcomes including clinical scores at 10 weeks and 12 months, cerebrospinal fluid data and patient-reported outcome measures. Patients receiving 6 weeks doxycycline reported slightly more side effects in week 5. Conclusion Our results strongly indicate that there are no benefits of doxycycline treatment beyond 2 weeks in European LNB.publishedVersio

Improved analysis of bacterial CGH data beyond the log-ratio paradigm

<p>Abstract</p> <p>Background</p> <p>Existing methods for analyzing bacterial CGH data from two-color arrays are based on log-ratios only, a paradigm inherited from expression studies. We propose an alternative approach, where microarray signals are used in a different way and sequence identity is predicted using a supervised learning approach.</p> <p>Results</p> <p>A data set containing 32 hybridizations of sequenced versus sequenced genomes have been used to test and compare methods. A ROC-analysis has been performed to illustrate the ability to rank probes with respect to Present/Absent calls. Classification into Present and Absent is compared with that of a gaussian mixture model.</p> <p>Conclusion</p> <p>The results indicate our proposed method is an improvement of existing methods with respect to ranking and classification of probes, especially for multi-genome arrays.</p

Assessment of cognitive function, structural brain changes and fatigue 6 months after treatment of neuroborreliosis

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Adaptation of Brucella melitensis Antimicrobial Susceptibility Testing to the ISO 20776 Standard and Validation of the Method

This article belongs to the Special Issue Emerging Themes in Brucella and Brucellosis.Brucellosis, mainly caused by Brucella (B.) melitensis, is associated with a risk of chronification and relapses. Antimicrobial susceptibility testing (AST) standards for B. melitensis are not available, and the agent is not yet listed in the EUCAST breakpoint tables. CLSI recommendations for B. melitensis exist, but they do not fulfill the requirements of the ISO 20776 standard regarding the culture medium and the incubation conditions. Under the third EU Health Programme, laboratories specializing in the diagnostics of highly pathogenic bacteria in their respective countries formed a working group within a Joint Action aiming to develop a suitable method for the AST of B. melitensis. Under the supervision of EUCAST representatives, this working group adapted the CLSI M45 document to the ISO 20776 standard after testing and validation. These adaptations included the comparison of various culture media, culture conditions and AST methods. A Standard Operation Procedure was derived and an interlaboratory validation was performed in order to evaluate the method. The results showed pros and cons for both of the two methods but also indicate that it is not necessary to abandon Mueller–Hinton without additives for the AST of B. melitensis.This research was funded by the EU Health Programme 2014–2020, through the Consumers, Health, Agriculture and Food Executive Agency (CHAFEA, European Commission), the Joint Action EMERGE (CHAFEA n° 677 066) and the Joint Action SHARP (848096-SHARP JA).info:eu-repo/semantics/publishedVersio

Comparative Genomic Analysis of Pathogenic and Probiotic Enterococcus faecalis Isolates, and Their Transcriptional Responses to Growth in Human Urine

Urinary tract infection (UTI) is the most common infection caused by enterococci, and Enterococcus faecalis accounts for the majority of enterococcal infections. Although a number of virulence related traits have been established, no comprehensive genomic or transcriptomic studies have been conducted to investigate how to distinguish pathogenic from non-pathogenic E. faecalis in their ability to cause UTI. In order to identify potential genetic traits or gene regulatory features that distinguish pathogenic from non-pathogenic E. faecalis with respect to UTI, we have performed comparative genomic analysis, and investigated growth capacity and transcriptome profiling in human urine in vitro. Six strains of different origins were cultivated and all grew readily in human urine. The three strains chosen for transcriptional analysis showed an overall similar response with respect to energy and nitrogen metabolism, stress mechanism, cell envelope modifications, and trace metal acquisition. Our results suggest that citrate and aspartate are significant for growth of E. faecalis in human urine, and manganese appear to be a limiting factor. The majority of virulence factors were either not differentially regulated or down-regulated. Notably, a significant up-regulation of genes involved in biofilm formation was observed. Strains from different origins have similar capacity to grow in human urine. The overall similar transcriptional responses between the two pathogenic and the probiotic strain suggest that the pathogenic potential of a certain E. faecalis strain may to a great extent be determined by presence of fitness and virulence factors, rather than the level of expression of such traits