Evolutionary dynamics of methicillin-resistant Staphylococcus aureus within a healthcare system

BackgroundIn the past decade, several countries have seen gradual replacement of endemic multi-resistant healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) with clones that are more susceptible to antibiotic treatment. One example is Singapore, where MRSA ST239, the dominant clone since molecular profiling of MRSA began in the mid-1980s, has been replaced by ST22 isolates belonging to EMRSA-15, a recently emerged pandemic lineage originating from Europe.ResultsWe investigated the population structure of MRSA in Singaporean hospitals spanning three decades, using whole genome sequencing. Applying Bayesian phylogenetic methods we report that prior to the introduction of ST22, the ST239 MRSA population in Singapore originated from multiple introductions from the surrounding region; it was frequently transferred within the healthcare system resulting in a heterogeneous hospital population. Following the introduction of ST22 around the beginning of the millennium, this clone spread rapidly through Singaporean hospitals, supplanting the endemic ST239 population. Coalescent analysis revealed that although the genetic diversity of ST239 initially decreased as ST22 became more dominant, from 2007 onwards the genetic diversity of ST239 began to increase once more, which was not associated with the emergence of a sub-clone of ST239. Comparative genomic analysis of the accessory genome of the extant ST239 population identified that the Arginine Catabolic Mobile Element arose multiple times, thereby introducing genes associated with enhanced skin colonization into this population.ConclusionsOur results clearly demonstrate that, alongside clinical practice and antibiotic usage, competition between clones also has an important role in driving the evolution of nosocomial pathogen populations.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0643-z) contains supplementary material, which is available to authorized users.     

Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.

Results

Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage φ-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage.

Conclusions

The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1599-9) contains supplementary material, which is available to authorized users.     

Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo

The innate immune system relies to a great deal on the interaction of pattern recognition receptors with pathogen- or damage-associated molecular pattern molecules. Extracellular histones belong to the latter group and their release has been described to contribute to the induction of systemic inflammatory reactions. However, little is known about their functions in the early immune response to an invading pathogen. Here we show that extracellular histones specifically target monocytes in human blood and this evokes the mobilization of the chemotactic chemokines CXCL9 and CXCL10 from these cells. The chemokine induction involves the toll-like receptor 4/myeloid differentiation factor 2 complex on monocytes, and is under the control of interferon-γ. Consequently, subcutaneous challenge with extracellular histones results in elevated levels of CXCL10 in a murine air pouch model and an influx of leukocytes to the site of injection in a TLR4 dependent manner. When analyzing tissue biopsies from patients with necrotizing fasciitis caused by Streptococcus pyogenes, extracellular histone H4 and CXCL10 are immunostained in necrotic, but not healthy tissue. Collectively, these results show for the first time that extracellular histones have an important function as chemoattractants as their local release triggers the recruitment of immune cells to the site of infection.     

Incorporating environmental change over succession in an integral projection model of population dynamics of a forest herb

Despite seemingly obvious effects of environmental drivers, mechanisms behind long‐term changes in plant population sizes over time are often poorly known. We investigated how soil potassium concentration and seed predation are likely to change over time as a result of succession from deciduous forest to spruce forest, and how this affects population trajectories of Actaea spicata. Observations and addition experiments showed that high soil potassium concentration increased individual growth rates. Among‐site comparisons showed that soil potassium concentration was lower where proportion spruce was higher. Incorporation of a gradual increase in spruce over time in an integral projection model where individual growth depended on potassium suggested a net decrease in A. spicata population sizes over forest succession. This result suggests that small changes in factors with small effects on individual performance can influence patterns of species occupancy along successional gradients. We incorporated also density independent and density dependent effects of pre‐dispersal seed predation over succession into the same model. Seed predation influenced the tree composition at which A. spicata population growth was positive. However, significant effects of A. spicata population size on seed predation intensity did not translate into important feedback effects on population growth trajectories over succession. Our results illustrate how demographic models can be used to gain understanding of the mechanisms behind effects of environmental change on species abundances and distributions by the simultaneous inclusion of changing abiotic and biotic factors.     

Difference in extracellular radical release after chemotactic factor and calcium ionophore activation of the oxygen radical-generating system in human neutrophils

Results obtained with the luminol-dependent chemiluminescence technique show that with this technique, generation of radicals from an extra- as well as from an intracellular source is quantified. By means of a chemiluminescence technique, using human neutrophils stimulated with the chemoattractant formylmethionylleucylphenylalanine and the calcium ionophore ionomycin, two different mechanisms of radical production and release are demonstrated. The chemoattractant causes the cells to produce oxygen radicals which to a large extent are released from the cells. The calcium ionophore is also capable of stimulating radical formation but does not suffice for extracellular release. Furthermore, the removal of extracellular Ca2+ is of minor importance for the extracellular radical production, whereas it totally inhibits the generation of radicals with an intracellular localization. The mechanism(s) behind intracellular and extracellular production of oxygen radicals is discussed.     

Quantification of annexin I in subcellular fractions of human neutrophils reveals an exclusive cytosolic localisation

Annexin I is an abundant cytosolic protein in human neutrophils. Besides its intracellular location, annexin I is found as an extracellular protein and the pathway for secretion has been of interest since the protein lacks a signal sequence for secretion. It was recently shown that annexin I is stored in the secretory gelatinase granules of human neutrophils, suggesting that the protein might be released through a granule mobilisation and fusion process resembling classical secretion. In this study we have determined the intracellular localisation of annexin I in human neutrophils using subcellular fractionation, protein separation by SDS-PAGE and immunoblotting, and show that virtually all annexin I is localised in the cell cytosol.     

Characterization of a major histocompatibility class II A gene (Clha-DAA) with an embedded microsatellite marker in Atlantic herring (Clupea harengus L.)

An Atlantic herring major histocompatibility class II A ( Clha-DAA ) cDNA sequence has been characterized and was shown to encode a leader peptide, alpha-1 domain, alpha-2 domain, connecting peptide, transmembrane and cytoplasmic region. The Clha-DAA protein sequence has all the characteristics of a teleost class II A protein with conserved cysteines in both the alpha-1 and the alpha-2 domains and two potential N-linked glycosylation sites. Exon 2 sequences encoding the polymorphic alpha-1 domain from different individuals were analysed and revealed the presence of at least two loci. The Clha-DAA gene consists of four exons and three short introns. Four unique intron 3 sequences from multiple individuals were obtained and were shown to contain a (TG)_n microsatellite sequence. Primers were optimized such that only a single microsatellite locus designated Clha-DAA-INTR3 was amplified. Four herring populations from the North Sea and the Baltic Sea were genotyped for Clha-DAA-INTR3 . In total, 16 Clha-DAA-INTR3 alleles were detected; the distribution of the alleles showed no deviation from Hardy–Weinberg expectation. Levels of genetic differentiation among samples were of similar magnitude as have been reported earlier for neutral microsatellite loci between northern North Sea and Baltic Sea herring populations.