Faculty Bibliography

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.

The genome of Staphylococcus aureus has rapidly become one the most frequently sequenced among bacteria, with more than 40000 genome sequences uploaded to public databases. Computational resources required for analysis and quality assessment have lagged behind accumulation of sequence data. Improved analytic pipelines, in combination with the development of customized S. aureus reference databases, can be used to inform S. aureus biology and potentially predict clinical outcome. Here, we review the currently available data about S. aureus genome in public databases, and discuss their potential utility for understanding S. aureus evolution. Also discussed are ways to overcome challenges to the application of whole-genome sequencing data for prevention and management of S. aureus disease.

The recent spread of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) has brought increasing concerns of heightened disease severity and persistence following invasive disease. In line with the need for new treatment paradigms, two recent reports have shown that antibody-based therapies can restrict acute S. aureus infection and persistence and improve pathological symptoms.

Background. We have recently observed an increase in community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections among pediatric patients from Brooklyn hospitalized at a university-based teaching hospital in New York City. We performed a prospective study to determine the colonization prevalence of CA-MRSA among hospital admission, genome sequence strains causing infection and identified risk factors associated with CA-MRSA carriage in this population. Methods. Colonization data were obtained from routine infection control screening upon admission to the general pediatric and intensive care units. We used a questionnaire to identify risk factors for MRSA transmission. Additionally, single patient isolates of CA-MRSA were collected from the clinical microbiology laboratory. Medical record information was used to ascertain patient infection or colonization and to confirm community onset. Children from high-risk communities were identified via zip codes. Figure. Phylogenetic tree of clinical MRSA USA300 isolates from children living in high-risk zip codes (red), adult and pediatric patients at NYU Tisch Hospital (Blue), and USA300 Strains from around the United States (Green; Pfizer). Results. Children from the high-risk zip codes were 3 times as likely to be colonized with MRSA (9% versus 3% [p = 0.04]). No difference in methicillin-susceptible S. aureus colonization prevalence was observed between children from high-risk and low-risk communities. Likewise, the MRSA infection rate per 1000 patient days was 36 for children from high-risk zip codes, and 3.9 in children from low-risk zip codes (p < 0.0001). All isolates from patients in high risk zip codes analyzed to date belong to genotype USA300, the predominant CA-MRSA clone in the United States. Phylogenetic analyses suggest that these strains arose from expansion of an USA300 CAMRSA subclone. Potential risk factors for MRSA infection are being explored in conjunction with public health and community leaders. Conclusion. We identified a cluster of CA-MRSA strain USA300 among pediatric patients in a high risk Brooklyn community. Additional genomic comparisons and epidemiological data will be used to inform interventions and interrupt transmission. (Figure Presented)

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction.

The current study identified bacterial factors that may improve management of methicillin-resistant Staphylococcus aureus (MRSA) nosocomial pneumonia. Isolates were obtained from 386 patients enrolled in a randomized, controlled study of antibiotic efficacy. Isolates were screened for production of virulence factors and for vancomycin susceptibility. After adjustment for host factors such as severity of illness and treatment modality, cytotoxic activity was strongly and inversely associated with mortality; however, it had no effect on clinical cure. Isolates having low cytotoxicity, which were derived largely from healthcare-associated clones, exhibited a greater prevalence of vancomycin heteroresistance, and they were recovered more often from patients who were older and frailer. Additionally, a clone with low cytotoxic activity was associated with death and poor clinical improvement. Clone specificity and attenuated virulence appear to be associated with outcome. To our knowledge, these are the first correlations between MRSA virulence and mortality in nosocomial pneumonia.

BACKGROUND: Previous studies have demonstrated an association between antibiotic use and the development of skin abscesses. We tested the hypothesis that alterations in the composition of the cutaneous microbiota may predispose individuals to skin abscesses. METHODS: We studied 25 patients with skin abscesses and 25 age-matched controls, who each completed a questionnaire. Skin swab samples were obtained for DNA analysis from 4 sites around the abscess site (hereafter, "peri-abscess specimens") and from similar sites on the patient's contralateral side and on healthy control subjects. DNA was extracted and analyzed by quantitative polymerase chain reaction (qPCR) and high-throughput sequencing. The purulent abscess drainage was sent for culture. RESULTS: Fifteen patients with abscess were infected with Staphylococcus aureus. Use of nuc qPCR to quantitate S. aureus revealed a significantly greater frequency of positive results for peri-abscess and contralateral skin samples, compared with control skin specimens. Analysis of community structure showed greater heterogeneity in the control samples than in the peri-abscess and contralateral samples. Metagenomic analysis detected significantly more predicted genes related to metabolic activity in the peri-abscess specimens than in the control samples. CONCLUSIONS: The peri-abscess microbiome was similar to the contralateral microbiome, but both microbiomes differed from that for control patients. Host characteristics affecting microbial populations might be important determinants of abscess risk.