Automated categorization of methicillin-resistant Staphylococcus aureus clinical isolates into different clonal complexes by MALDI-TOF mass spectrometry.

Early identification of methicillin-resistant Staphylococcus aureus (MRSA) dominant clones involved in infection and initiation of adequate infection control measures are essential to limit MRSA spread and understand MRSA population dynamics. In this study we evaluated the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) for the automated discrimination of the major MRSA lineages (clonal complexes, CC) identified in our hospital during a 20-year period (1990-2009). A collection of 82 well-characterized MRSA isolates belonging to the four main CCs (CC5, CC8, CC22 and CC398) was split into a reference set (n = 36) and a validation set (n = 46) to generate pattern recognition models using the ClinProTools software for the identification of MALDI-TOF/MS biomarker peaks. The supervised neural network (SNN) model showed the best performance compared with two other models, with sensitivity and specificity values of 100% and 99.11%, respectively. Eleven peaks (m/z range: 3278-6592) with the highest separation power were identified and used to differentiate all four CCs. Validation of the SNN model using ClinProTools resulted in a positive predictive value (PPV) of 99.6%. The specific contribution of each peak to the model was used to generate subtyping reference signatures for automated subtyping using the BioTyper software, which successfully classified MRSA isolates into their corresponding CCs with a PPV of 98.9%. In conclusion, we find this novel automated MALDI-TOF/MS approach to be a promising, powerful and reliable tool for S. aureus typing.

Lack of association between genotypes and haematogenous seeding infections in a large cohort of patients with methicillin-resistant Staphylococcus aureus bacteraemia from 21 Spanish hospitals.

There is increasing concern regarding the association between certain methicillin-resistant Staphylococcus aureus (MRSA) genotypes and poor clinical outcome. To assess this issue, a large cohort of 579 subjects with MRSA bacteraemia was prospectively followed from June 2008 to December 2009, in 21 hospitals in Spain. Epidemiology, clinical data, therapy, and outcome were recorded. All MRSA strains were analysed in a central laboratory. Presence of a haematogenous seeding infection was the dependent variable in an adjusted logistic regression model. Of the 579 patients included in the study, 84 (15%) had haematogenous seeding infections. Microdilution vancomycin median MIC (IQR) was 0.73 (0.38-3) mg/L. Most MRSA isolates (n = 371; 67%) belonged to Clonal Complex 5 (CC5) and carried an SCCmec element type IV and agr type 2. Isolates belonging to ST8-agr1-SCCmecIV, ST22-agr1-SCCmecIV and ST228-agr2-SCCmecI--a single locus variant of ST5--accounted for 8%, 9% and 9% of the isolates, respectively. After adjusting by clinical variables, any of the clones was associated with increased risk of haematogenous seeding infections. Higher vancomycin MIC was not identified as an independent risk factor, either. In contrast, persistent bacteraemia (OR 4.2; 2.3-7.8) and non-nosocomial acquisition (3.0; 1.7-5.6) were associated with increased risk.

Predictive factors for early mortality among patients with methicillin-resistant Staphylococcus aureus bacteraemia.

OBJECTIVES: A high proportion of patients with methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia die within a few days of the onset of infection. However, predictive factors for early mortality (EM) have barely been examined. The aim of this study was to determine the predictive factors for EM in patients with MRSA bacteraemia. METHODS: All episodes of MRSA bacteraemia were prospectively followed in 21 Spanish hospitals from June 2008 to December 2009. Epidemiology, clinical data, therapy and outcome were recorded. All MRSA strains were analysed in a central laboratory. Mortality was defined as death from any cause occurring in the 30 days after the onset of MRSA bacteraemia. EM was defined as patients who died within the first 2 days, and late mortality (LM) for patients who died after this period. Multivariate analyses were performed by using logistic regression models. RESULTS: A total of 579 episodes were recorded. Mortality was observed in 179 patients (31%): it was early in 49 (8.5%) patients and late in 130 (22.5%). Independent risk factors for EM were [OR (95% CI)] initial Pitt score >3 [3.99 (1.72-3.24)], previous rapid fatal disease [3.67 (1.32-10.24)], source of infection lower respiratory tract or unknown [3.76 (1.31-10.83) and 2.83 (1.11-7.21)], non-nosocomial acquisition [2.59 (1.16-5.77)] and inappropriate initial antibiotic therapy [3.59 (1.63-7.89)]. When predictive factors for EM and LM were compared, inappropriate initial antibiotic therapy was the only distinctive predictor of EM, while endocarditis and lower respiratory tract sources both predicted LM. CONCLUSIONS: In our large cohort of patients several factors were related to EM, but the only distinctive predictor of EM was inappropriate initial antibiotic therapy.

Predictive factors for mortality in patients with methicillin-resistant Staphylococcus aureus bloodstream infection: impact on outcome of host, microorganism and therapy.

Mortality related to methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection (BSI) remains high, despite changes in the epidemiology. To analyze the current predictive factors for mortality we conducted a prospective study in a large cohort of patients with MRSA-BSI from 21 Spanish hospitals. Epidemiology, clinical data, therapy and outcome were recorded. All MRSA strains were analysed, including susceptibility to antibiotics and molecular characterization. Vancomycin MICs (V-MIC) were tested by the E-test and microdilution methods. Time until death was the dependent variable in a Cox regression analysis. Overall, 579 episodes were included. Acquisition was nosocomial in 59% and vascular catheter was the most frequent source (38%). A dominant PFGE genotype was found in 368 (67%) isolates, which belonged to Clonal Complex (CC)5 and carried SCCmecIV and agr2. Microdilution V-MIC50 and V-MIC90 were 0.7 and 1.0 mg/L, respectively. Initial therapy was appropriate in 66% of episodes. Overall mortality was observed in 179 (32%) episodes. The Cox-regression analysis identified age >70 years (HR 1.88), previous fatal disease (HR 2.16), Pitt score >1 (HR 3.45), high-risk source (HR 1.85) and inappropriate initial treatment (HR 1.39) as independent predictive factors for mortality. CC5 and CC22 (HR 0.52 and 0.45) were associated with significantly lower mortality rates than CC8. V-MIC ≥1.5 did not have a significant impact on mortality, regardless of the method used to assess it.

Methicillin-susceptible Staphylococcus aureus clone related to the early pandemic phage type 80/81 causing an outbreak among residents of three occupational centres in Barcelona, Spain.

In the 1950s an unusually virulent and transmissible penicillin-resistant Staphylococcus aureus clone harbouring Panton-Valentine leukocidin (PVL) genes, known as phage type 80/81 and subsequently identified as multilocus sequence type (ST) 30, emerged and caused serious infections in hospitals and the community. We describe an outbreak of skin infections caused by a PVL-positive, methicillin-susceptible S. aureus (MSSA) strain of ST1472, related to phage type 80/81, in three associated occupational centres. After identification of the first patient an active case-finding strategy was initiated among the three centres. Epidemiological and clinical features were indistinguishable from outbreaks currently caused by community-acquired methicillin-resistant S. aureus. The S. aureus was cultured and identified from nasal swabs and skin lesions by conventional methods; PVL was detected using a PCR assay. Pulsed-field gel electrophoresis and DNA-array-based genotyping were applied to MSSA isolates. MSSA was identified in nasal swabs from 49 of 133 individuals (37%). A single pulsed-field gel electrophoresis pattern, belonging to ST1472 (CC30) and PVL positivity, were detected in 20 individuals, including eight of 18 skin cultures, i.e. 15% of the screened individuals were colonized by the epidemic strain. Nasal and cutaneous decontamination with 5% nasal mupirocin ointment and 2% aqueous chlorhexidine was implemented for all individuals. Patients with active skin infections were treated with a first-generation cephalosporin. General recommendations were made to prevent cross-transmission. No new cases were reported over the following 90 days.