Faculty Bibliography

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.

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.

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.

BACKGROUND: European studies suggest that living near high-density livestock production increases the risk of sequence type (ST) 398 methicillin-resistant Staphylococcus aureus (MRSA) colonization. To our knowledge, no studies have evaluated associations between livestock production and human infection by other strain types. OBJECTIVES: We evaluated associations between MRSA molecular subgroups and high-density livestock production. METHODS: We conducted a yearlong 2012 prospective study on a stratified random sample of patients with culture-confirmed MRSA infection; we oversampled patients from the Geisinger Health System with exposure to high-density livestock production in Pennsylvania. Isolates were characterized using S. aureus protein A (spa) typing and detection of Panton-Valentine leukocidin (PVL) and scn genes. We compared patients with one of two specific MRSA strains with patients with all other strains of MRSA isolates, using logistic regression that accounted for the sampling design, for two different exposure models: one based on the location of the animals (livestock model) and the other on crop field application of manure (crop field model). RESULTS: Of 196 MRSA isolates, we identified 30 spa types, 47 PVL-negative and 15 scn-negative isolates, and no ST398 MRSA. Compared with quartiles 1-3 combined, the highest quartiles of swine livestock and dairy/veal crop field exposures were positively associated with community-onset-PVL-negative MRSA (CO-PVL-negative MRSA vs. all other MRSA), with adjusted odds ratios of 4.24 (95% CI: 1.60, 11.25) and 4.88 (95% CI: 1.40, 17.00), respectively. The association with CO-PVL-negative MRSA infection increased across quartiles of dairy/veal livestock exposure (trend p = 0.05). CONCLUSIONS: Our findings suggest that other MRSA strains, beyond ST398, may be involved in livestock-associated MRSA infection in the United States.