Faculty Bibliography

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.

A total of 434 methicillin-resistant Staphylococcus aureus (MRSA) baseline isolates were collected from subjects enrolled in a prospective, double-blind randomized trial comparing linezolid versus vancomycin for the treatment of nosocomial pneumonia. Isolates were susceptibility tested by broth microdilution, examined for inducible clindamycin resistance by D-test, and screened for heterogeneous resistance to vancomycin (hVISA) by the Etest macromethod. All strains were subjected to Panton-Valentine leukocidin (PVL) screening, and SCCmec, pulsed-field gel electrophoresis (PFGE), and spa typing. Selected strains were evaluated by multilocus sequence typing (MLST). Clonal complexes (CCs) were assigned based on the spa and/or MLST results. Most strains were CC5 (56.0%), which originated from North America (United States) (CC5-MRSA-SCCmec II/IV; 70.0%), Asia (CC5-MRSA-II; 14.0%) and Latin America (CC5-MRSA-I/II; 12.3%). The second- and third-most-prevalent clones were CC8-MRSA-IV (23.3%) and CC239-MRSA-III (11.3%), respectively. Furthermore, the CC5-MRSA-I/II clone predominated in Asia (50.7% within this region) and Latin America (66.7%), followed by CC239-MRSA-III (32.8% and 28.9%, respectively). The European strains were CC8-MRSA-IV (34.5%), CC22-MRSA-IV (18.2%), or CC5-MRSA-I/II/IV (16.4%), while the U.S. MRSA isolates were CC5-MRSA-II/IV (64.4%) or CC8-MRSA-IV (28.8%). Among the U.S. CC8-MRSA-II/IV strains, 73.7% (56/76 [21.2% of all U.S. MRSA strains]) clustered within USA300. One strain from the United States (USA800) was intermediate to vancomycin (MIC, 4 mug/ml). All remaining strains were susceptible to linezolid, daptomycin, vancomycin, and teicoplanin. hVISA strains (14.5%) were predominantly CC5-MRSA-II, from South Korea, and belonged to a single PFGE type. Overall, each region had two predominant clones. The USA300 rate corroborates previous reports describing increased prevalence of USA300 strains causing invasive infections. The prevalence of hVISA was elevated in Asia, and these strains were associated with CC5.

Inactivating mutations in the Staphylococcus aureus virulence regulator agr are associated with worse outcomes in bacteremic patients. However, whether agr dysfunction is primarily a cause or a consequence of early bacteremia is unknown. Analysis of 158 paired S. aureus clones from blood and nasal carriage sites in individual patients revealed that recovery of an agr-defective mutant from blood was usually predicted by the agr functionality of carriage isolates. Many agr-positive blood isolates produced low levels of hemolytic toxins, but levels were similar to those of colonizing strains within patients, suggesting that introduction into the blood did not select for mutations with minor functional effects. Evidently, the transition from commensalism to opportunism in S. aureus does not require full virulence in hospitalized patients. Furthermore, agr-defective mutants were found in uninfected nasal carriers in the same proportion as in carriers who develop bacteremia, suggesting low correlation between virulence and infectivity.

Staphylococcus aureus infections are a significant cause of morbidity and mortality in health care settings. S. aureus clinical isolates vary in the function of the accessory gene regulator (agr), which governs the expression of virulence determinants, including surface and exoproteins, while agr activity has been correlated with patient outcome and treatment efficiency. Here we describe a duplex real-time nucleic acid sequence-based amplification (NASBA) detection and quantification platform for rapid determination of agr functionality in clinical isolates. Using the effector of agr response, RNAIII, as the assay target, and expression of the gyrase gene (gyrB) as a normalizer, we were able to accurately discriminate agr functionality in a single reaction. Time to positivity (TTP) ratios between gyrB and RNAIII showed very good correlation with the ratios of RNAIII versus gyrB RNA standard inputs and were therefore used as a simple readout to evaluate agr functionality. We validated the assay by characterizing 106 clinical S. aureus isolates, including strains with genetically characterized agr mutations. All isolates with dysfunctional agr activity exhibited a TTP ratio (TTP(gyrB)/TTP(RNAIII)) lower than 1.10, whereas agr-positive isolates had a TTP ratio higher than this value. The results showed that the assay was capable of determining target RNA ratios over 8 logs (10(-3) to 10(4)) with high sensitivity and specificity, suggesting the duplex NASBA assay may be useful for rapid determination of agr phenotypes and virulence potential in S. aureus clinical isolates.

Bloodstream infection with Staphylococcus aureus is common and can be fatal. However, virulence factors that contribute to lethality in S. aureus bloodstream infection are poorly defined. We discovered that LukED, a commonly overlooked leucotoxin, is critical for S. aureus bloodstream infection in mice. We also determined that LukED promotes S. aureus replication in vivo by directly killing phagocytes recruited to sites of haematogenously seeded tissue. Furthermore, we established that murine neutrophils are the primary target of LukED, as the greater virulence of wild-type S. aureus compared with a lukED mutant was abrogated by depleting neutrophils. The in vivo toxicity of LukED towards murine phagocytes is unique among S. aureus leucotoxins, implying its crucial role in pathogenesis. Moreover, the tropism of LukED for murine phagocytes highlights the utility of murine models to study LukED pathobiology, including development and testing of strategies to inhibit toxin activity and control bacterial infection