Faculty Bibliography

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.

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.

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.

Staphylococcus aureus is one of the most successful bacterial pathogens, harboring a vast repertoire of virulence factors in its arsenal. As such, the genetic manipulation of S. aureus chromosomal DNA is an important tool for the study of genes involved in virulence and survival in the host. Previously reported allelic exchange vectors for S. aureus are shuttle vectors that can be propagated in Escherichia coli, so that standard genetic manipulations can be carried out. Most of the vectors currently in use carry the temperature-sensitive replicon (pE194ts) that was originally developed for use in Bacillus subtilis. Here we show that in S. aureus, the thermosensitivity of a pE194ts vector is incomplete at standard non-permissive temperatures (42 degrees C), and replication of the plasmid is impaired but not abolished. We report rpsL-based counterselection vectors, with an improved temperature-sensitive replicon (pT181 repC3) that is completely blocked for replication in S. aureus at non-permissive and standard growth temperature (37 degrees C). We also describe a set of temperature-sensitive vectors that can be cured at standard growth temperature. These vectors provide highly effective tools for rapidly generating allelic replacement mutations and curing expression plasmids, and expand the genetic tool set available for the study of S. aureus.

Mutations that alter virulence and antibiotic susceptibility arise and persist during Staphylococcus aureus bacteremia. However, an experimental system demonstrating transmission following bacteremia has been lacking, and thus implications of within-host adaptation for between-host transmission are unknown. We report that S. aureus disseminates to the gastrointestinal tract of mice following intravenous injection and readily transmits to cohoused naive mice. Both intestinal dissemination and transmission were linked to the production of virulence factors based on gene deletion studies of the sae and agr two-component systems. Furthermore, antimicrobial selection for antibiotic-resistant S. aureus displaced susceptible S. aureus from the intestine of infected hosts, which led to the preferential transmission and dominance of antibiotic-resistant bacteria among cohoused untreated mice. These findings establish an animal model to investigate gastrointestinal dissemination and transmission of S. aureus and suggest that adaptation during the course of systemic infection has implications beyond the level of a single host.

Donor-derived bacterial infection is a recognized complication of solid organ transplantation (SOT). The present report describes the clinical details and successful outcome in a liver transplant recipient despite transmission of methicillin-resistant Staphylococcus aureus (MRSA) from a deceased donor with MRSA endocarditis and bacteremia. We further describe whole genome sequencing (WGS) and complete de novo assembly of the donor and recipient MRSA isolate genomes, which confirms that both isolates are genetically 100% identical. We propose that similar application of WGS techniques to future investigations of donor bacterial transmission would strengthen the definition of proven bacterial transmission in SOT, particularly in the presence of highly clonal bacteria such as MRSA. WGS will further improve our understanding of the epidemiology of bacterial transmission in SOT and the risk of adverse patient outcomes when it occurs.