Faculty Bibliography

BACKGROUND:Respiratory viral infections are a leading cause of disease worldwide. However, the overall community prevalence of infections has not been properly assessed, as standard surveillance is typically acquired passively among individuals seeking clinical care. METHODS:We conducted a prospective cohort study in which participants provided daily diaries and weekly nasopharyngeal specimens that were tested for respiratory viruses. These data were used to analyze healthcare seeking behavior, compared with cross-sectional ED data and NYC surveillance reports, and used to evaluate biases of medically attended ILI as signal for population respiratory disease and infection. RESULTS:The likelihood of seeking medical attention was virus-dependent: higher for influenza and metapneumovirus (19%-20%), lower for coronavirus and RSV (4%), and 71% of individuals with self-reported ILI did not seek care and half of medically attended symptomatic manifestations did not meet the criteria for ILI. Only 5% of cohort respiratory virus infections and 21% of influenza infections were medically attended and classifiable as ILI. We estimated 1 ILI event per person/year but multiple respiratory infections per year. CONCLUSION/CONCLUSIONS:Standard, healthcare-based respiratory surveillance has multiple limitations. Specifically, ILI is an incomplete metric for quantifying respiratory disease, viral respiratory infection, and influenza infection. The prevalence of respiratory viruses, as reported by standard, healthcare-based surveillance, is skewed toward viruses producing more severe symptoms. Active, longitudinal studies are a helpful supplement to standard surveillance, can improve understanding of the overall circulation and burden of respiratory viruses, and can aid development of more robust measures for controlling the spread of these pathogens.

Staphylococcus aureus infection is a major public health threat in part due to the spread of antibiotic resistance and repeated failures to develop a protective vaccine. Infection is associated with production of virulence factors that include exotoxins that attack host barriers and cellular defenses, such as the leukocidin (Luk) family of bicomponent pore-forming toxins. To investigate the structural basis of antibody-mediated functional inactivation of Luk toxins, we generated a panel of murine monoclonal antibodies (MAbs) that neutralize host cell killing by the γ-hemolysin HlgCB. By biopanning these MAbs against a phage-display library of random Luk peptide fragments, we identified a small subregion within the rim domain of HlgC as the epitope for all the MAbs. Within the native holotoxin, this subregion folds into a conserved β-hairpin structure, with exposed key residues, His252 and Tyr253, required for antibody binding. On the basis of the phage-display results and molecular modeling, a 15-amino-acid synthetic peptide representing the minimal epitope on HlgC (HlgC241-255) was designed, and preincubation with this peptide blocked antibody-mediated HIgCB neutralization. Immunization of mice with HlgC241-255 or the homologous LukS246-260 subregion peptide elicited serum antibodies that specifically recognized the native holotoxin subunits. Furthermore, serum IgG from patients who were convalescent for invasive S. aureus infection showed neutralization of HlgCB toxin activity ex vivo, which recognized the immunodominant HlgC241-255 peptide and was dependent on His252 and Tyr253 residues. We have thus validated an efficient, rapid, and scalable experimental workflow for identification of immunodominant and immunogenic leukotoxin-neutralizing B-cell epitopes that can be exploited for new S. aureus-protective vaccines and immunotherapies.

Free-living mammals, such as humans and wild mice, display heightened immune activation compared with artificially maintained laboratory mice. These differences are partially attributed to microbial exposure as laboratory mice infected with pathogens exhibit immune profiles more closely resembling that of free-living animals. Here, we examine how colonization by microorganisms within the natural environment contributes to immune system maturation by releasing inbred laboratory mice into an outdoor enclosure. In addition to enhancing differentiation of T cell populations previously associated with pathogen exposure, outdoor release increased circulating granulocytes. However, these "rewilded" mice were not infected by pathogens previously implicated in immune activation. Rather, immune system changes were associated with altered microbiota composition with notable increases in intestinal fungi. Fungi isolated from rewilded mice were sufficient in increasing circulating granulocytes. These findings establish a model to investigate how the natural environment impacts immune development and show that sustained fungal exposure impacts granulocyte numbers.

Unbiased identification of individual, immunogenic B-cell epitopes in major antigens of a pathogen remains a technology challenge for vaccine discovery. We therefore developed a platform for rapid phage display screening of deep recombinant libraries consisting of as little as a single major pathogen antigen. Using the bi-component pore-forming leukocidin (Luks) exotoxins of the major pathogen Staphylococcus aureus (Sa) as a prototype, we randomly fragmented and separately ligated the Hemolysin gamma A (HlgA) and LukS genes into a custom-built, phage-display system, termed pComb-Opti8. Deep sequence analysis of barcoded amplimers of the HlgA and LukS gene fragment libraries demonstrated that biopannng against a cross-reactive anti-Luk mAb recovered convergent molecular clones with short overlapping homologous sequences. We thereby identified an 11-amino acid sequence that is highly conserved in four Luk toxin subunits, and is ubiquitous in representation within Sa clinical isolates. The isolated 11-amino acid peptide probe was predicted to retain the native 3D-conformation seen within the Luk holotoxin. Indeed, this peptide was recognized by the selecting anti-Luk mAb, and using mutated peptides we showed that a particular amino acid side-chain was essential for these interactions. Furthermore, murine immunization with this peptide elicited IgG-responses that were highly reactive with both the autologous synthetic peptide and the full-length Luk toxin homologues. Thus, using a gene fragment, phage-display based pipeline, we have identified and validated immunogenic B-cell epitopes that are cross-reactive between members of the pore-forming leukocidin family. This approach could be harnessed to identify novel epitopes for a much needed Sa-protective subunit vaccine.

Background. Methicillin-resistant staphylococcus aureus (MRSA) colonization of hospitalized patients is associated with higher readmission rates and increased morbidity. Depending on the mechanisms of transmission, numerous potential control interventions exist to reduce the burden of disease. However, given the preponderance of asymptomatic colonization, it is challenging to quantify the relative importance of different transmission mechanisms and assess control efficacy. By identifying clusters of transmission, whole-genome sequencing (WGS) provides an opportunity to overcome these challenges. Methods. We sought to apply cluster analysis techniques to WGS data for MRSA, in order to assess MRSA prevalence, transmissibility, the degree of transmission heterogeneity and the potential effectiveness of control. Our model builds upon previous work that showed a direct relationship between the size distribution of infection clusters, the effective reproduction number (R) and the dispersion parameter (k). To demonstrate its functionality, our model was applied to existing WGS data for MRSA isolates collected during a 12 month period in the East of England (DOI: 10.1126/scitranslmed.aak9745) Results. The effective reproduction number for the East of England data is 0.29 (95% CI: 0.24-0.36). The dispersion parameter is 0.09 (0.03-0.33) reflecting a high degree of transmission heterogeneity. This implies all transmission is caused by just 12% of the cases. Targeted control of these cases could have decreased overall burden of MRSA colonization by 29% during the time period of the study. Conclusion. The high degree of transmission heterogeneity seen in MRSA transmission suggests that the risk for infection is variable.This observation motivates the need for more detailed mechanistic modeling of hospital-based MRSA transmission that integrates patients-specific factors, movement data and genome sequencing. Such models could be used to forecast which patients are at greatest risk for either acquiring or transmitting MRSA, thereby improving targeted control

Background. Colonization with Staphylococcus aureus, particularly MRSA, is a crucial risk factor for subsequent infection. Decolonization measures are often undertaken to prevent recurrent MRSA infection and transmission; however, increasing rate of resistance to the gold standard mupirocin has been noted globally. At our institution, there is >85% high-level resistance to mupirocin among strains from a geographically defined genotypic cluster of CA-MRSA in children from Orthodox communities in Brooklyn. Retapamulin is a topical bacteriostatic pleuromutilin antibiotic that has demonstrated excellent in vitro activity against mupirocin-resistant isolates from pediatric patients with MRSA infection presenting to our institution suggesting that it may be a promising alternative decolonization therapy. We sought to determine the efficacy of retapamulin as a topical decolonizing agent against mupirocin-resistant MRSA among the identified high-risk Brooklyn cluster via a randomized, placebo-controlled, double-blinded phase three trial. Methods. Children aged 9 months-17 years who resided in high-risk zip codes used as a proxy for Orthodox Jewish predominant neighborhoods were recruited either from inpatient units at NYU Langone or at a partnered community clinic. Participants were screened via nasal and rectal culture to detect MRSA colonization. Enrolled participants were randomized to receive either retapamulin or placebo and instructed to apply the ointment nasally and rectally twice a day for 5 days. Repeat nasal and rectal swab cultures were collected one week and one month after completion of topical therapy to assess MRSA colonization status. The change in colonization rates was assessed via Fisher's exact test. Results. 173 participants were screened from December 2017 to March 2019 in which 47 ultimately underwent randomization (23 in the retapamulin group and 24 in the placebo group). The median age was 3.9 years (SD 3.5 years). Children in the placebo group were 15.2 times more likely to be colonized with MRSA after one week of the decolonization protocol compared with the retapamulin group (OR 15.2, CI 2.8-81, P = 0.0004). However, children in the placebo group were only 1.1 times more likely to be colonized with MRSA after one month compared with the retapamulin group (OR 1.1, CI 0.3-3.9, P = 1). (*Full data analysis currently in progress with additional results available soon.) Conclusion. In this small pilot randomized trial, children who received retapamulin had a significantly lower rate of MRSA colonization and higher rates of clearance compared with placebo at one week post decolonization, but no significant difference at the one month mark. These data suggest that retapamulin is a promising alternative short-term nasal and peri-rectal decolonzing therapy in order to prevent infections and the spread of this mupirocin-resistant MRSA clone among pediatric patients in this affected community and our hospital

The pathogen Staphylococcus aureus colonizes and infects a variety of different sites within the human body. To adapt to these different environments, S. aureus relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in S. aureus remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator purR resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the purR mutant was still highly virulent in the presence of mutations that disrupt PurR's canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of purR Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that S. aureus toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the purR mutant. Thus, S. aureus repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis.

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.