Anita Blomfeldt

Genotyping of 353 Staphylococcus aureus Bloodstream Isolates Collected between 2004 and 2009 at a Norwegian University Hospital and Potential Associations with Clinical Parameters

ABSTRACT We analyzed 353 Staphylococcus aureus bloodstream isolates from 2004 to 2009 to identify dominant genotypes, changes over time, and associations between genotype, phenotype, and clinical parameters. The isolates were genotyped with regard to spa type and presence of Panton-Valentine leukocidin and toxic shock syndrome toxin 1-encoding genes. A high level of genetic diversity was detected. All but three isolates were methicillin sensitive. Interestingly, spa clonal complex 021 showed a weak association with higher all-cause hospital mortality.

Molecular Characterization of Methicillin-Sensitive Staphylococcus aureus Isolates from Bacteremic Patients in a Norwegian University Hospital

ABSTRACT Staphylococcus aureus bacteremia is common in both nosocomial and community settings, and the pathogenicity of the microbe depends upon a large repertoire of virulence factors. S. aureus bacteremia isolates (n = 126) were characterized using DNA microarrays. Clonal complexes 5, 8, 15, 30, and 45 accounted for 74.6% of the isolates. We identified geographical differences in dominating clones and toxin gene profiles. One isolate was methicillin resistant. Potential associations between age and genotype were detected.

Molecular characterisation of methicillin-sensitive Staphylococcus aureus from deep surgical site infections in orthopaedic patients

Staphylococcus aureus is a leading cause of surgical site infections (SSIs). The association between S. aureus genotypes and the severity of illness is, however, incompletely understood. The aim of the study was to genotype S. aureus isolates from deep SSI in orthopaedic patients to identify molecular markers associated with invasive S. aureus infections. DNA microarray analysis was performed on S. aureus isolates collected from 60 patients with deep SSI following major orthopaedic surgery, while 57 isolates from nasal carriers served as controls. Genes associated with antibiotic resistance, adhesion, immune evasion, tissue invasion and toxin production were detected. The bone sialoprotein-binding protein gene (bbp) was more frequent in isolates from SSI patients compared to nasal carriers (95.0% vs. 82.5%), suggesting a role in invasive disease. No major differences in other molecular virulence markers could be distinguished among isolates from the two clinical groups, suggesting that any S. aureus strain may cause invasive infection. Our study reveals important genotypic information on isolates obtained from deep SSI following orthopaedic procedures.

spa typing alone is not sufficient to demonstrate endemic establishment of meticillin-resistant Staphylococcus aureus in a low-prevalence country

BACKGROUND The prevalence of meticillin-resistant Staphylococcus aureus (MRSA) in Norway is low but increasing. Over the last decade, numerous nursing homes have experienced MRSA outbreaks. One genetic lineage, spa type t304, has been identified at multiple nursing homes and has caused large outbreaks lasting for several years. AIM To evaluate whether spa typing is sufficient for the detection of MRSA spread and endemic establishment in a low-prevalence area, using spa type t304 as the test organism. METHODS All spa type t304 isolates detected in 1991-2010 in the most densely populated area of Norway were included. Time and place of bacterial sampling were recorded. The isolates were analysed using multi-locus sequence typing, staphylococcal cassette chromosome mec typing, detection of lukS/F-PV and pulsed-field gel electrophoresis (PFGE). FINDINGS In total, 181 spa type t304 isolates were identified in three of 23 municipalities. Most (91%) of the isolates could be linked to 13 nursing homes, eight of which experienced outbreaks. PFGE analysis revealed three PFGE types, consisting of 19 PFGE patterns; 95% of the isolates were PFGE type 2. In total, PFGE types 2 and 3 accounted for 99% of all nursing home isolates, and included isolates from different nursing homes, different outbreaks and different time periods. Additional genetic analyses did not further differentiate between the spa type t304 isolates. CONCLUSION MRSA spa type t304 appears to have established itself as an endemic genetic lineage in the study area. spa typing does not provide sufficient resolution when investigating the spread of an endemic-like genetic lineage in a low-prevalence area, and should be supplemented by additional typing techniques.

DNA microarray analysis of Staphylococcus aureus causing bloodstream infection: bacterial genes associated with mortality?

Providing evidence for microbial genetic determinants’ impact on outcome in Staphylococcus aureus bloodstream infections (SABSI) is challenging due to the complex and dynamic microbe–host interaction. Our recent population-based prospective study reported an association between the S. aureus clonal complex (CC) 30 genotype and mortality in SABSI patients. This follow-up investigation aimed to examine the genetic profiles of the SABSI isolates and test the hypothesis that specific genetic characteristics in S. aureus are associated with mortality. SABSI isolates (n = 305) and S. aureus CC30 isolates from asymptomatic nasal carriers (n = 38) were characterised by DNA microarray analysis and spa typing. Fisher’s exact test, least absolute shrinkage and selection operator (LASSO) and elastic net regressions were performed to discern within four groups defined by patient outcome and characteristics. No specific S. aureus genetic determinants were found to be associated with mortality in SABSI patients. By applying LASSO and elastic net regressions, we found evidence suggesting that agrIII and cna were positively and setC (=selX) and seh were negatively associated with S. aureus CC30 versus non-CC30 isolates. The genes chp and sak, encoding immune evasion molecules, were found in higher frequencies in CC30 SABSI isolates compared to CC30 carrier isolates, indicating a higher virulence potential. In conclusion, no specific S. aureus genes were found to be associated with mortality by DNA microarray analysis and state-of-the-art statistical analyses. The next natural step is to test the hypothesis in larger samples with higher resolution methods, like whole genome sequencing.

Can MLVA Differentiate among Endemic-Like MRSA Isolates with Identical Spa-Type in a Low-Prevalence Region?

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Norway is low, but an endemic-like MRSA clone with Staphylococcal protein A (spa)-type t304 has been established especially in nursing homes in the Oslo region causing several large outbreaks. The challenge was that spa-typing and the gold standard Pulsed-Field Gel Electrophoresis (PFGE) were inadequate in discriminating isolates in outbreak investigations. Additional higher resolution genotyping methods were needed. The aims of this study were a) to evaluate whether Multiple-Locus Variable number of tandem repeat Analysis (MLVA) could differentiate within the PFGE clusters between epidemiologically related and unrelated endemic-like ST8-MRSA-IV-t304-PVL-neg (MRSA-t304) isolates and b) investigate the evolution of the endemic-like MRSA-t304 clone over a 15-year time period. All MRSA-t304 isolates detected in the region from 1998 through April 2013 were included. In total, 194 of 197 isolates were available for PFGE and MLVA analyses. PFGE results on isolates from 1998–2010 have been published previously. Two PFGE clusters subdivided into eight MLVA types were detected. One major outbreak clone (PFGE cluster C2/ MLVA type MT5045) appeared from 2004 to 2011 causing long-lasting and large outbreaks in seven nursing homes and one hospital. Five new MLVA types (N = 9 isolates) differing in only one VNTR compared to the outbreak clone C2/MT5045 were detected, but only one (C2/MT5044) was seen after 2011. We suggest that MLVA can replace PFGE analysis, but MLVA may not be the optimal method in this setting as it did not discriminate between all epidemiologically unrelated isolates. The results may indicate that all eight outbreaks in different locations within the PFGE C2 cluster may be branches of one large regional outbreak. The major outbreak strain C2/MT5045 may now, however, be under control, extinguished or has moved geographically.

No associations established between single nucleotide polymorphisms in human Toll-like receptor 2 and Toll-interacting protein and Staphylococcus aureus bloodstream infections

Staphylococcus aureus bloodstream infections (SABSI) are associated with high morbidity and mortality. The Toll‐like receptor 2 (TLR2) and Toll‐interacting protein (TOLLIP) are important in recognition and regulation of human innate immunity response to S. aureus. Single nucleotide polymorphisms (SNPs) in the TLR2 and TOLLIP encoding genes have been associated with disease, including BSI. The aim of this study was to examine potential associations between a selection of SNPs in the genes encoding TLR2 and TOLLIP, and predisposition, severity, and outcome of SABSI. All patients ≥18 years of age with at least one S. aureus positive blood culture collected from March 2011 through February 2014 at Akershus University Hospital, Lørenskog, Norway, were considered for inclusion. Patients attending elective orthopaedic surgery (total hip and knee replacements, lumbar surgery) served as a control group. The TLR2 Arg753Gln, TLR2 Pro631His, TOLLIP rs5743942, and rs5743867 polymorphisms were analysed using TaqMan SNP Genotyping Assays. A total of 209 SABSI patients and 295 controls were included. The TLR2 Arg753Gln and TLR2 Pro631His polymorphisms were infrequent with no homozygotes and <10% heterozygotes. The included TLR2 and TOLLIP polymorphisms were not associated with susceptibility to SABSI, severity, 30‐day all‐cause mortality, or SABSI caused by the clonal complex 30 (CC30) genotype.

Predictors of one-year all-cause mortality and infection-related mortality in patients with Staphylococcus aureus bacteraemia

Abstract Objectives: Staphylococcus aureus bacteraemia (SAB) is a common infection associated with significant short-term mortality. Little is known about long-term prognosis. The aim of this study was to determine one-year all-cause mortality and infection-related mortality and associated predictors. Methods: Data from 303 consecutive patients with SAB were prospectively collected from March 2011 to February 2014. All patients were followed one year or until death. Results: One-year all-cause- and infection-related mortality were 36.7% and 20.8%, respectively. For all-cause mortality, in multivariable logistic regression analysis, age 70–79 years (OR 3.9; 95% CI 1.7–9.1; p = .001), Charlson Comorbidity index ≥3 (OR 6.9; 95% CI 2.7–17.3; p < .001), healthcare-associated infection (OR 2.3; 95% CI 1.1–4.9; p = .03) and severe sepsis (OR 3.6; 95% CI 1.8–7.1; p < .001) were independent predictors of outcome. For infection-related mortality, the predictors were similar, except for healthcare-associated infection that lost significance. The vast majority (89%) of infection-related deaths occurred within 30 days. Conclusions: This study demonstrates additional significant all-cause mortality in patients with SAB beyond 30 days to one year, mainly driven by high age and comorbidity. As a result, SAB can be considered an indirect marker of high risk of death in these patients. Follow-up beyond 30 days does not add significant information with respect to infection-related mortality.

Correction for Blomfeldt et al., Molecular Characterization of Methicillin-Sensitive Staphylococcus aureus Isolates from Bacteremic Patients in a Norwegian University Hospital

Volume 51, no. 1, p. [345–347][1], 2013. Page 346, [Table 1][2]: Owing to misclassification of infections as hospital acquired (HA) or community acquired (CA), some entries in the third and fourth columns were wrong. The conclusions of the paper are unchanged, as the misclassifications went in

Correction for Aamot et al., Genotyping of 353 Staphylococcus aureus Bloodstream Isolates Collected between 2004 and 2009 at a Norwegian University Hospital and Potential Associations with Clinical Parameters

Volume 50, no. 9, p. [3111–3114][1], 2012. Page 3112, [Table 1][2]: For the sixth entry in the “CC” section of the first column, “ spa C304/024” should read “ spa CC304/024,” and owing to misclassification of infections as hospital acquired (HA) or community acquired (CA), several