Faculty Bibliography

BACKGROUND:Rheumatoid arthritis patients are at increased risk for periprosthetic joint infection after arthroplasty. The reason is multifactorial. Nasal colonization with Staphylococcus aureus is a modifiable risk factor; carriage rates in RA patients are unknown. The goal of this study is to determine the S aureus nasal carriage rates of RA patients on biologics, RA patients on traditional disease-modifying anti-rheumatic drugs (DMARDs), and osteoarthritis. METHODS:Consecutive patients with RA on biologics (±DMARDs), RA on non-biologic DMARDs, or OA were prospectively enrolled from April 2017 to May 2018. One hundred twenty-three patients were determined necessary per group to show a difference in carriage rates. Patients underwent a nasal swab and answered questions to identify additional risk factors. S aureus positive swabs were further categorized using spa typing. Logistic regression evaluated the association with S aureus colonization between the groups after controlling for known risk factors. RESULTS:RA patients on biologics, 70% of whom were on DMARDs, had statistically significant increase in S aureus colonization (37%) compared to RA on DMARDs alone (24%), or OA (20%) (P = .01 overall). After controlling for glucocorticoids, antibiotic use, recent hospitalization, and diabetes, RA on biologics had a significant increased risk of S aureus nasal colonization (Odds ratio 1.80, 95% confidence interval 1.00-3.22, P = .047). CONCLUSION/CONCLUSIONS:S aureus colonization risk was increased for RA on biologics compared to RA not on biologics and OA. Nasal S aureus carriage increases the risk of surgical site infection; this modifiable risk factor should be addressed prior to total joint arthroplasty for this higher risk patient group.

The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytes and lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB), are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstrate that LukED and HlgAB cause vascular congestion and derangements in vascular fluid distribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines (DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the critical target for lethality. Consistent with this, LukED and HlgAB injure primary human endothelial cells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells are resistant to toxin-mediated lethality. During bloodstream infection in mice, DARC targeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potential for S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulence factors.

The past two decades have witnessed an alarming expansion of staphylococcal disease caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The factors underlying the epidemic expansion of CA-MRSA lineages such as USA300, the predominant CA-MRSA clone in the United States, are largely unknown. Previously described virulence and antimicrobial resistance genes that promote the dissemination of CA-MRSA are carried by mobile genetic elements, including phages and plasmids. Here, we used high-resolution genomics and experimental infections to characterize the evolution of a USA300 variant plaguing a patient population at increased risk of infection to understand the mechanisms underlying the emergence of genetic elements that facilitate clonal spread of the pathogen. Genetic analyses provided conclusive evidence that fitness (manifest as emergence of a dominant clone) changed coincidently with the stepwise emergence of (i) a unique prophage and mutation of the regulator of the pyrimidine nucleotide biosynthetic operon that promoted abscess formation and colonization, respectively, thereby priming the clone for success; and (ii) a unique plasmid that conferred resistance to two topical microbiocides, mupirocin and chlorhexidine, frequently used for decolonization and infection prevention. The resistance plasmid evolved through successive incorporation of DNA elements from non-S. aureus spp. into an indigenous cryptic plasmid, suggesting a mechanism for interspecies genetic exchange that promotes antimicrobial resistance. Collectively, the data suggest that clonal spread in a vulnerable population resulted from extensive clinical intervention and intense selection pressure toward a pathogen lifestyle that involved the evolution of consequential mutations and mobile genetic elements.

Staphylococcus aureus is a human pathogen responsible for high morbidity and mortality worldwide. Recurrent infections with this bacterium are common, suggesting that S. aureus thwarts the development of sterilizing immunity. S. aureus strains that cause disease in humans produce up to five different bicomponent toxins (leukocidins) that target and lyse neutrophils, innate immune cells that represent the first line of defense against S. aureus infections. However, little is known about the role of leukocidins in blunting adaptive immunity. Here, we explored the effects of leukocidins on human dendritic cells (DCs), antigen-presenting cells required for the development of adaptive immunity. Using an ex vivo infection model of primary human monocyte-derived dendritic cells, we found that S. aureus, including strains from different clonal complexes and drug resistance profiles, effectively kills DCs despite efficient phagocytosis. Although all purified leukocidins could kill DCs, infections with live bacteria revealed that S. aureus targets and kills DCs primarily via the activity of leukocidin LukAB. Moreover, using coculture experiments performed with DCs and autologous CD4⁺ T lymphocytes, we found that LukAB inhibits DC-mediated activation and proliferation of primary human T cells. Taken together, the data determined in the study reveal a novel immunosuppressive strategy of S. aureus whereby the bacterium blunts the development of adaptive immunity via LukAB-mediated injury of DCs.IMPORTANCE Antigen-presenting cells such as dendritic cells (DCs) fulfill an indispensable role in the development of adaptive immunity by producing proinflammatory cytokines and presenting microbial antigens to lymphocytes to trigger a faster, specific, and long-lasting immune response. Here, we studied the effect of Staphylococcus aureus toxins on human DCs. We discovered that the leukocidin LukAB hinders the development of adaptive immunity by targeting human DCs. The ability of S. aureus to blunt the function of DCs could help explain the high frequency of recurrent S. aureus infections. Taken together, the results from this study suggest that therapeutically targeting the S. aureus leukocidins may boost effective innate and adaptive immune responses by protecting innate leukocytes, enabling proper antigen presentation and T cell activation.

Background. Controlling methicillin-resistant Staphylococcus aureus (MRSA) colonization is a common strategy to prevent transmission and recurrent infection. Standard decolonization regimens include nasal application of mupirocin ointment; however, increasing rates of mupirocin-resistance (Mup-R) have been noted globally. At our institution there has been an increase in community-acquired MRSA (CA-MRSA) infections among children living in Brooklyn, New York. A genotypic geographic cluster of an outbreak clone of the CA-MRSA strain USA 300 with a high rate (>85%) of mupirocin resistance, mediated by the plasmid borne mupA gene, was identified prompting investigation into an alternative decolonizing agent. We sought to investigate retapamulin, a topical pleuromutilin antibiotic, which has been shown to be effective against S. aureus with in vitro and in vivo activity against MRSA and a low propensity to develop resistance. Methods. Broth microdilution was used to determine the minimum inhibitory concentrations (MIC) of retapamulin against 53 Mup-R MRSA isolates collected from pediatric patients (aged 9 months-17 years) presenting to our institution over an 18 month period with clinical MRSA infection. Susceptibility defined as <=0.5 mg/L susceptible (EUCAST). Whole genome sequence data were analyzed for the presence of rplC and cfr gene mutations known to confer resistance to retapamulin. Results. All 53 isolates were susceptible to retapamulin. 49/53 (92%) strains were inhibited at MIC 0.25 mg/L, 2/53 (4%) at MIC 0.125 mg/L, and 2/53 (4%) at MIC 0.5 mg/L. DNA sequence analysis showed that one isolate had a first-step mutation in the rplC gene, but it was not associated with reduced phenotypic susceptibility to retapamulin, as the MIC of that isolate was 0.25 mg/L. Conclusion. Retapamulin demonstrated excellent in vitro activity against a genotypic cluster of Mup-R isolates from pediatric patients presenting to our institution with MRSA infection. These data suggest that retapamulin may be a promising alternative decolonization therapy for MRSA and a viable option to prevent the spread of mupirocin-resistant MRSA clones. Further research includes an ongoing randomized, placebo-controlled trial testing the in vivo efficacy of retapamulin as a nasal and perirectal decolonizing agent in children

Therapy for bacteremia caused by Staphylococcus aureus is often ineffective, even when treatment conditions are optimal according to experimental protocols. Adapted subclones, such as those bearing mutations that attenuate agr-mediated virulence activation, are associated with persistent infection and patient mortality. To identify additional alterations in agr-defective mutants, we sequenced and assembled the complete genomes of clone pairs from colonizing and infected sites of several patients in whom S. aureus demonstrated a within-host loss of agr function. We report that events associated with agr inactivation result in agr-defective blood and nares strain pairs that are enriched in mutations compared to pairs from wild-type controls. The random distribution of mutations between colonizing and infecting strains from the same patient, and between strains from different patients, suggests that much of the genetic complexity of agr-defective strains result from prolonged infection or therapy-induced stress. However, in one of the agr-defective infecting strains, multiple genetic changes resulted in increased virulence in a murine model of bloodstream infection, bypassing the mutation of agr and raising the possibility that some changes were selected. Expression profiling correlated the elevated virulence of this agr-defective mutant to restored expression of the agr-regulated ESAT6-like Type VII secretion system, a known virulence factor. Thus, additional mutations outside the agr locus can contribute to diversification and adaptation during infection by S. aureusagr mutants associated with poor patient outcome.

Staphylococcus aureus is an opportunistic pathogen that causes a range of serious infections associated with significant morbidity, by strains increasingly resistant to antibiotics. However, to date all candidate vaccines have failed to induce protective immune responses in humans. We need a more comprehensive understanding of the antigenic targets important in the context of human infection. To investigate infection-associated immune responses, patients were sampled at initial presentation and during convalescence from three types of clinical infection; skin and soft tissue infection (SSTI), prosthetic joint infection (PJI) and pediatric hematogenous osteomyelitis (PHO). Reactivity of serum IgG was tested with an array of recombinant proteins, representing over 2,652 in-vitro-translated open reading frames (ORFs) from a community-acquired methicillin-resistant S. aureus USA300 strain. High-level reactivity was demonstrated for 104 proteins with serum IgG in all patient samples. Overall, high-level IgG-reactivity was most commonly directed against a subset of secreted proteins. Although based on limited surveys, we found subsets of S. aureus proteins with differential reactivity with serum samples from patients with different clinical syndromes. Together, our studies have revealed a hierarchy within the diverse proteins of the S. aureus "immunome", which will help to advance efforts to develop protective immunotherapeutic agents.

Invasive community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections disproportionately affect children, but there are few pediatric reports of pericarditis and mediastinitis caused by CA-MRSA in previously healthy children. Here we report a severe case of CA-MRSA pericarditis with extension to the mediastinum and carotid sheath in a previously healthy 8-month-old infant who was successfully treated with surgical interventions and with a combination of daptomycin and vancomycin. The relatively indolent clinical course in this patient was notable given the significant extent of infection. This case highlights the potential virulence of CA-MRSA in previously healthy children and the importance of early diagnosis, prompt drainage, and appropriate antibiotic coverage.