Bo Shopsin

Prevalence of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus in the Community

Recent reports indicate that community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections are increasing and may now involve persons without risk factors predisposing for acquisition. To estimate the extent of community MRSA in New York City, the prevalence of S. aureus and MRSA nasal colonization in a well-patient population of 500 children and guardians was determined. The prevalence of S. aureus nasal carriage was 35% for children and 28% for guardians. One person with predisposing risk factors was colonized with an MRSA, which was identified as the predominant clone found in New York City hospitals. A high degree of methicillin-susceptible S. aureus strain diversity was noted, with no apparent selection for specific clonal types. Thus, MRSA colonization is not ubiquitous in persons without predisposing risk outside of the health care environment. Bacterial competition and a lack of strong selection may limit the community spread of MRSA and can account for its sporadic distribution.

agr function in clinical Staphylococcus aureus isolates

The accessory gene regulator (agr) of Staphylococcus aureus is a global regulator of the staphylococcal virulon, which includes secreted virulence factors and surface proteins. The agr locus is important for virulence in a variety of animal models of infection, and has been assumed by inference to have a major role in human infection. Although most human clinical S. aureus isolates are agr(+), there have been several reports of agr-defective mutants isolated from infected patients. Since it is well known that the agr locus is genetically labile in vitro, we have addressed the question of whether the reported agr-defective mutants were involved in the infection or could have arisen during post-isolation handling. We obtained a series of new staphylococcal isolates from local clinical infections and handled these with special care to avoid post-isolation mutations. Among these isolates, we found a number of strains with non-haemolytic phenotypes owing to mutations in the agr locus, and others with mutations elsewhere. We have also obtained isolates in which the population was continuously heterogeneous with respect to agr functionality, with agr(+) and agr(-) variants having otherwise indistinguishable chromosomal backgrounds. This finding suggested that the agr(-) variants arose by mutation during the course of the infection. Our results indicate that while most clinical isolates are haemolytic and agr(+), non-haemolytic and agr(-) strains are found in S. aureus infections, and that agr(+) and agr(-) variants may have a cooperative interaction in certain types of infections.

Prevalence of agr Specificity Groups among Staphylococcus aureus Strains Colonizing Children and Their Guardians

ABSTRACT PCR-based assays were used to evaluate agr locus nucleotide polymorphism for the identification of agr autoinducer receptor specificity groups within a population of Staphylococcus aureus isolates colonizing children and their guardians. All isolates could be assigned to one of three major agr groups that had similar prevalences, regardless of whether isolates were implicated in transmission of S. aureus within families. Among healthy carriers, agr groups I to III appear to be equally fit, which may reflect selection for the coexistence of S. aureus strains in a population.

Characterization of a new cytotoxin that contributes to Staphylococcus aureus pathogenesis

Staphylococcus aureus is an important pathogen that continues to be a significant global health threat because of the prevalence of methicillin‐resistant S. aureus strains (MRSA). The pathogenesis of this organism is partly attributed to the production of a large repertoire of cytotoxins that target and kill innate immune cells, which provide the first line of defence against S. aureus infection. Here we demonstrate that leukocidin A/B (LukAB) is required and sufficient for the ability of S. aureus, including MRSA, to kill human neutrophils, macrophages and dendritic cells. LukAB targets the plasma membrane of host cells resulting in cellular swelling and subsequent cell death. We found that S. aureus lacking lukAB are severely impaired in their ability to kill phagocytes during bacteria–phagocyte interaction, which in turn renders the lukAB‐negative staphylococci more susceptible to killing by neutrophils. Notably, we show that lukAB is expressed in vivo within abscesses in a murine infection model and that it contributes significantly to pathogenesis of MRSA in an animal host. Collectively, these results extend our understanding of how S. aureus avoids phagocyte‐mediated clearance, and underscore LukAB as an important factor that contributes to staphylococcal pathogenesis.

Staphylococcus aureus leucocidin ED contributes to systemic infection by targeting neutrophils and promoting bacterial growth in vivo

Bloodstream infection with Staphylococcus aureus is common and can be fatal. However, virulence factors that contribute to lethality in S. aureus bloodstream infection are poorly defined. We discovered that LukED, a commonly overlooked leucotoxin, is critical for S. aureus bloodstream infection in mice. We also determined that LukED promotes S. aureus replication in vivo by directly killing phagocytes recruited to sites of haematogenously seeded tissue. Furthermore, we established that murine neutrophils are the primary target of LukED, as the greater virulence of wild‐type S. aureus compared with a lukED mutant was abrogated by depleting neutrophils. The in vivo toxicity of LukED towards murine phagocytes is unique among S. aureus leucotoxins, implying its crucial role in pathogenesis. Moreover, the tropism of LukED for murine phagocytes highlights the utility of murine models to study LukED pathobiology, including development and testing of strategies to inhibit toxin activity and control bacterial infection.

Prevalence of agr Dysfunction among Colonizing Staphylococcus aureus Strains

Mutations in the staphylococcal virulence regulator gene agr frequently occur during Staphylococcus aureus infection. Whether agr-defective strains are fit for colonization, an important prerequisite for infection, is unknown. Screening by means of assays to detect delta-hemolysin activity and agr autoinducing peptide production indicated that 15 ( approximately 9%) of 160 healthy human subjects were colonized with an agr-defective strain or a mixture of agr-positive and -defective S. aureus strains. The presence of identical agr-defective strains in family members suggests that these strains are transmissible. Additionally, carriage of an agr-defective strain was associated with hospitalization, raising the possibility that such strains may be selected in a nosocomial setting.

Mutations in agr Do Not Persist in Natural Populations of Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus organisms vary in the function of the staphylococcal virulence regulator gene agr. To test for a relationship between agr and transmission in S. aureus, we determined the prevalence and genetic basis of agr dysfunction among nosocomial methicillin-resistant S. aureus (MRSA) in an area of MRSA endemicity. Identical inactivating agr mutations were not detected in epidemiologically unlinked clones within or between hospitals. Additionally, most agr mutants had single mutations, indicating that they were short lived. Collectively, the results suggest that agr dysfunction is adaptive for survival in the infected host but that it may be counteradaptive outside infected host tissues.

The in Vitro Generation of Post-Golgi Vesicles Carrying Viral Envelope Glycoproteins Requires an ARF-like GTP-binding Protein and a Protein Kinase C Associated with the Golgi Apparatus*

We have developed a system that recreates in vitro the generation of post-Golgi vesicles from an isolated Golgi fraction prepared from vesicular stomatitis virus- or influenza virus-infected Madin-Darby canine kidney or HepG2 cells. In this system, vesicle generation is temperature- and ATP-dependent and requires a supply of cytosolic proteins, including an N-ethylmaleimide-sensitive factor distinct from NSF. Cytosolic proteins obtained from yeast were as effective as mammalian cytosolic proteins in supporting vesicle formation and had the same requirements. The vesicles produced (50-80 nm in diameter) are depleted of the trans Golgi marker sialyltransferase, contain the viral glycoprotein molecules with their cytoplasmic tails exposed, and do not show an easily recognizable protein coat. Vesicle generation was inhibited by brefeldin A, which indicates that it requires the activation of an Arf-like GTP-binding protein that promotes assembly of a vesicle coat. Vesicles formed in the presence of the nonhydrolyzable GTP analogue guanosine 5′-3-O-(thio)triphosphate retained a nonclathrin protein coat resembling that of COP-coated vesicles, and sedimented more rapidly in a sucrose gradient than the uncoated ones generated in its absence. This indicates that GTP hydrolysis is not required for vesicle generation but that it is for vesicle uncoating. The activity of a Golgi-associated protein kinase C (PKC) was found to be necessary for the release of post-Golgi vesicles, as indicated by the capacity of a variety of inhibitors and antibodies to PKC to suppress it, as well as by the stimulatory effect of the PKC activator 12-O-tetradecanoylphorbol-13-acetate.

Preventing Surgical Site Infections: A Randomized, Open-Label Trial of Nasal Mupirocin Ointment and Nasal Povidone-Iodine Solution

BACKGROUND Treatment of Staphylococcus aureus colonization before surgery reduces risk of surgical site infection (SSI). The regimen of nasal mupirocin ointment and topical chlorhexidine gluconate is effective, but cost and patient compliance may be a barrier. Nasal povidone-iodine solution may provide an alternative to mupirocin. METHODS We conducted an investigator-initiated, open-label, randomized trial comparing SSI after arthroplasty or spine fusion in patients receiving topical chlorhexidine wipes in combination with either twice daily application of nasal mupirocin ointment during the 5 days before surgery or 2 applications of povidone-iodine solution into each nostril within 2 hours of surgical incision. The primary study end point was deep SSI within the 3 months after surgery. RESULTS In the modified intent-to-treat analysis, a deep SSI developed after 14 of 855 surgical procedures in the mupirocin group and 6 of 842 surgical procedures in the povidone-iodine group (P = .1); S. aureus deep SSI developed after 5 surgical procedures in the mupirocin group and 1 surgical procedure in the povidone-iodine group (P = .2). In the per protocol analysis, S. aureus deep SSI developed in 5 of 763 surgical procedures in the mupirocin group and 0 of 776 surgical procedures in the povidone-iodine group (P = .03). CONCLUSIONS Nasal povidone-iodine may be considered as an alternative to mupirocin in a multifaceted approach to reduce SSI. TRIAL REGISTRATION ClinicalTrials.gov identifier: NCT01313182.

Evolution of hypervirulence by a MRSA clone through acquisition of a transposable element.

Staphylococcus aureus has evolved as a pathogen that causes a range of diseases in humans. There are two dominant modes of evolution thought to explain most of the virulence differences between strains. First, virulence genes may be acquired from other organisms. Second, mutations may cause changes in the regulation and expression of genes. Here we describe an evolutionary event in which transposition of an IS element has a direct impact on virulence gene regulation resulting in hypervirulence. Whole‐genome analysis of a methicillin‐resistant S. aureus (MRSA) strain USA500 revealed acquisition of a transposable element (IS256) that is absent from close relatives of this strain. Of the multiple copies of IS256 found in the USA500 genome, one was inserted in the promoter sequence of repressor of toxins (Rot), a master transcriptional regulator responsible for the expression of virulence factors in S. aureus. We show that insertion into the rot promoter by IS256 results in the derepression of cytotoxin expression and increased virulence. Taken together, this work provides new insight into evolutionary strategies by which S. aureus is able to modify its virulence properties and demonstrates a novel mechanism by which horizontal gene transfer directly impacts virulence through altering toxin regulation.