Genetic Variation in Spatio-Temporal Confined USA300 Community-Associated MRSA Isolates: A Shift from Clonal Dispersion to Genetic Evolution?

NTRODUCTION: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are increasingly isolated, with USA300-0114 being the predominant clone in the USA. Comparative whole genome sequencing of USA300 isolates collected in 2002, 2003 and 2005 showed a limited number of single nucleotide polymorphisms and regions of difference. This suggests that USA300 has undergone rapid clonal expansion without great genomic diversification. However, whole genome comparison of CA-MRSA has been limited to isolates belonging to USA300. The aim of this study was to compare the genetic repertoire of different CA-MRSA clones with that of HA-MRSA from the USA and Europe through comparative genomic hybridization (CGH) to identify genetic clues that may explain the successful and rapid emergence of CA-MRSA. MATERIALS AND METHODS: Hierarchical clustering based on CGH of 48 MRSA isolates from the community and nosocomial infections from Europe and the USA revealed dispersed clustering of the 19 CA-MRSA isolates. This means that these 19 CA-MRSA isolates do not share a unique genetic make-up. Only the PVL genes were commonly present in all CA-MRSA isolates. However, 10 genes were variably present among 14 USA300 isolates. Most of these genes were present on mobile elements. CONCLUSION: The genetic variation present among the 14 USA300 isolates is remarkable considering the fact that the isolates were recovered within one month and originated from a confined geographic area, suggesting continuous evolution of this clone

Comparison of the identification of coagulase-negative staphylococci by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tuf sequencing

The increasing incidence of coagulase-negative staphylococci (CoNS) in hospital-acquired infections underlines the need for an accurate and simple identification of Staphylococcus isolates at the species level. Sequencing of the tuf gene has been shown to be the most accurate for the species identification of CoNS. We determined the species of 62 consecutive clinical and 31 reference CoNS isolates by tuf gene sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Species assignment by MALDI-TOF-MS and tuf sequencing was congruent in all cases. We conclude that MALDI-TOF-MS is accurate for identifying CoNS in routine clinical practice. The study also identified an unexpectedly high number of cases of Staphylococcus capitis infections among 62 consecutive CoNS isolates in 2009 at the University Medical Center Utrecht, the Netherlands

A genomic approach to explore the development and evolution of methicillin-resistant staphylococci

The aim of this thesis was to investigate genomic differences between CA-MRSA and HA-MRSA to provide our understanding of the factors favouring the recent emergence of CA-MRSA. Since MRSA may result from de novo acquisition of SCCmec by methicillin-susceptible isolates the potential role of methicillin-resistant CoNS as a source of SCCmec for these MRSA strains was also assessed. The first part of this thesis focuses on genomic differences between CA-MRSA and HA-MRSA and within these groups. For this purpose we compared the genetic repertoire of different CA-MRSA clones to that of HA-MRSA from the USA and Europe through comparative genomic hybridization (CGH). In chapter two new methods to analyze microarrays were investigated, in particular when a reference signal is absent for a number of probes on the microarray. These methods were used for identifying genetic differences between and within HA- and CA-MRSA isolates. Comparative genomic hybridization results from HA- and CA-MRSA isolates obtained with this microarray showed unexpected genetic differences among 14 USA300 CA-MRSA isolates. The 14 CA-MRSA isolates were recovered within one month and originated from a confined geographic area (Chicago) (chapter three). The last chapter of the first part, chapter four, describes the results of whole genome sequencing of the first ST80 CA-MRSA isolate from the USA, and its comparison to the European clone. The second part focuses on the epidemiology of SCCmec in staphylococci. CoNS are thought to be the potential reservoir of the methicillin resistance determinant mecA for S. aureus. The aim was to assess the potential role of CoNS as a reservoir of SCCmec elements for MRSA. First we evaluated a reliable and easy CoNS species determination method (MALDI-TOF MS), (chapter five). Oliveira et al. showed that DNA sequencing of an internal fragment of ccrB enables the characterization of the ccrAB allotype present in MRSA strains and, after cluster analyses, the prediction of SCCmec types I-IV and VI. In chapter six we determined the genetic relatedness of ccrB sequences of a large, diverse, Europe-wide collection of mecA-positive CoNS with ccrB sequences from MRSA isolates In this thesis the evolutionary development of methicillin-resistant staphylococci was explored by comparative genomics and molecular epidemiology of the Staphylococcal Cassette Chromosome encoding methicillin resistance (SCCmec). This knowledge is important in order to reconstruct the evolutionary past of specific resistant staphylococcal clones which may help us to understand and possibly predict the success of these clones in the future. This information may direct infection prevention measures and ways to treat infections caused by these staphylococci

Interleukin-1 receptor antagonist anakinra as treatment for paradoxical responses in HIV-negative tuberculosis patients: A case series

BACKGROUND: Paradoxical inflammatory responses can occur during microbiologically successful antituberculous therapy. Optimal treatment is unknown, but corticosteroids are used most often. It is likely that interleukin-1 (IL-1) plays a central role in the development of these paradoxical responses, and if corticosteroids fail or are undesirable because of adverse effects, anti-IL-1 therapy may therefore be a rational choice. METHODS: We present seven HIV-negative tuberculosis patients with paradoxical responses, two with exclusively pulmonary and five with extrapulmonary tuberculosis. All had received corticosteroids, with unsatisfactory effect. Patients were treated with the IL-1 receptor antagonist anakinra and monitored for reduction of fever and inflammatory markers, imaging evidence of stabilization or regression of lesions, and respiratory improvement. FINDINGS: Six patients had anemia and four patients had lymphopenia at the start of the antituberculosis treatment. Fever was present in six patients at the moment of paradoxical response. Anakinra resulted in the decrease of fever within days, followed by resolution of symptoms and radiological improvement in five patients. Anakinra induced neutropenia, necessitating its cessation in two patients, who recovered quickly afterward. CONCLUSION: Anakinra can be considered in HIV-negative tuberculosis patients with paradoxical responses when steroids fail or are undesired. Given its favorable safety profile and reversible side effects, it is conceivable that anakinra might also be used as first-line adjuvant treatment for paradoxical responses. FUNDING: A.v.L. and R.v.C. are supported by National Institutes of Health (R01AI145781)