Identification of Small Molecules Exhibiting Oxacillin Synergy through a Novel Assay for Inhibition of vraTSR Expression in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) strains that are resistant to all forms of penicillin have become an increasingly common and urgent problem threatening human health. They are responsible for a wide variety of infectious diseases ranging from minor skin abscesses to life-threatening severe infections. The vra operon that is conserved among S. aureus strains encodes a three-component signal transduction system (vraTSR) that is responsible for sensing and responding to cell wall stress. We developed a novel and multifaceted assay to identify compounds that potentiate the activity of oxacillin, essentially restoring efficacy of oxacillin against MRSA, and performed high-throughput screening (HTS) to identify oxacillin potentiators. HTS of 13,840 small-molecule compounds from an antimicrobial-focused Life Chemicals library, using the MRSA cell-based assay, identified three different inhibitor scaffolds. Checkerboard assays for synergy with oxacillin, reverse transcriptase PCR (RT-PCR) assays against vraR expression, and direct confirmation of interaction with VraS by surface plasmon resonance (SPR) further verified them to be viable hit compounds. A subsequent structure-activity relationship (SAR) study of the best scaffold with diverse analogs was utilized to improve potency and provides a strong foundation for further development.

Retrospective Identification of a Broad IgG Repertoire Differentiating Patients With S. aureus Skin and Soft Tissue Infections From Controls.

Background: Although the relevance of humoral immunity for protection against S. aureus skin and soft tissue infections (SSTIs) has been suggested by several animal and human studies, the question of which human antibodies may be protective has so far impeded the development of a safe and effective vaccine. Because most adults have developed certain anti-S. aureus antibodies due to S. aureus colonization or infection, we hypothesized that the titers of antibodies to S. aureus in uninfected controls would differ from those in infected patients and would also differ in infected patients from the time of acute infection to a 40-day convalescent serum. Methods: To test these hypotheses, we measured human antibody levels against a panel of 134 unique antigens comprising the S. aureus surfome and secretome in subjects with active culture-confirmed S. aureus SSTIs (cases) and in controls with no infection, using a novel S. aureus protein microarray. Results: Most S. aureus SSTI patients (n = 60) and controls (n = 142) had antibodies to many of the tested S. aureus antigens. Univariate analysis showed statistically weak differences in the IgG levels to some antigens in the SSTI patient (case) sera compared with controls. Antibody levels to most tested antigens did not increase comparing acute with 40-day serum. Multiple logistic regression identified a rich subset of antigens that, by their antibody levels, together correctly differentiated all cases from all controls. Conclusions: Antibodies directed against S. aureus antigens were present both in patients with S. aureus SSTIs and in uninfected control patients. We found that SSTI patients and controls could be distinguished only based on differences in antibody levels to many staphylococcal surface and secreted antigens. Our results demonstrate that in the studied population, the levels of anti-S. aureus antibodies appear largely fixed, suggesting that there may be some level of unresponsiveness to natural infection.

Decreasing Incidence of Skin and Soft-tissue Infections in 86 US Emergency Departments, 2009-2014.

Background: The incidence of skin and soft-tissue infections (SSTIs), for which human immunodeficiency virus (HIV) is a significant risk factor, in United States emergency departments (EDs) increased dramatically after 2000 with the emergence of community-associated methicillin-resistant Staphylococcus aureus. Few studies have examined SSTI incidence among HIV-infected and non-HIV-infected patients in the United States after 2010. Methods: Data were obtained for patient encounters at all academic medical center EDs affiliated with the Vizient clinical data warehouse assigned an SSTI-associated code based on the International Classification of Diseases, Ninth Revision, between 1 January 2009 and 31 December 2014. The rate was calculated per 1000 ED encounters by year and stratified by SSTI, HIV infection, or both, and by age group, race, payer type, and region of care. Poisson regression was used to assess temporal change over the study period. Results: In 2009-2014, a total of 47317 HIV-associated and 820440 SSTI-associated encounters were recorded among 25239781 ED patient encounters. The rate of SSTIs decreased by 8% among all patients and by 14.6%, among those with HIV infection. The SSTI incidence overall decreased from 32.0 to 29.7 per 1000 ED encounters between 2009 and 2014. HIV-infected patients had a significantly higher rate of SSTIs than HIV-uninfected patients (adjusted rate ratio, 1.91; 95% confidence interval, 1.84-1.99). Conclusions: The decline in SSTI incidence in US EDs between 2009 and 2014 is a remarkable epidemiologic shift from the increase in SSTIs after 2000, and further research is necessary to assess reasons for this decrease.

Distinct T-helper cell responses to Staphylococcus aureus bacteremia reflect immunologic comorbidities and correlate with mortality.

BACKGROUND: The dysregulated host immune response that defines sepsis varies as a function of both the immune status of the host and the distinct nature of the pathogen. The degree to which immunocompromising comorbidities or immunosuppressive medications affect the immune response to infection is poorly understood because these patients are often excluded from studies about septic immunity. The objectives of this study were to determine the immune response to a single pathogen (Staphylococcus aureus) among a diverse case mix of patients and to determine whether comorbidities affect immune and clinical outcomes. METHODS: Blood samples were drawn from 95 adult inpatients at multiple time points after the first positive S. aureus blood culture. Cox proportional hazards modeling was used to determine the associations between admission neutrophil counts, admission lymphocyte counts, cytokine levels, and 90-day mortality. A nested case-control flow cytometric analysis was conducted to determine T-helper type 1 (Th1), Th2, Th17, and regulatory T-cell (Treg) subsets among a subgroup of 28 patients. In a secondary analysis, we categorized patients as either having immunocompromising disorders (human immunodeficiency virus and hematologic malignancies), receiving immunosuppressive medications, or being not immunocompromised. RESULTS: Higher neutrophil-to-lymphocyte count ratios and higher Th17 cytokine responses relative to Th1 cytokine responses early after infection were independently associated with mortality and did not depend on the immune state of the patient (HR 1.93, 95% CI 1.17-3.17, p = 0.01; and HR 1.13, 95% CI 1.01-1.27, p = 0.03, respectively). On the basis of flow cytometric analysis of CD4 T-helper subsets, an increasing Th17/Treg response over the course of the infection was most strongly associated with increased mortality (HR 4.41, 95% CI 1.69-11.5, p < 0.01). This type of immune response was most common among patients who were not immunocompromised. In contrast, among immunocompromised patients who died, a decreasing Th1/Treg response was most common. CONCLUSIONS: The association of both increased Th17 responses and increased neutrophil counts relative to lymphocyte counts with mortality suggests that an overwhelming inflammatory response is detrimental. However, the differential responses of patients according to immune state suggest that immune status is an important clinical indicator that should be accounted for in the management of septic patients, as well as in the development of novel immunomodulatory therapies.

Can Methicillin-resistant Staphylococcus aureus Silently Travel From the Gut to the Wound and Cause Postoperative Infection? Modeling the "Trojan Horse Hypothesis".

OBJECTIVE: To determine whether intestinal colonization with methicillin-resistant Staphylococcus aureus (MRSA) can be the source of surgical site infections (SSIs). BACKGROUND: We hypothesized that gut-derived MRSA may cause SSIs via mechanisms in which circulating immune cells scavenge MRSA from the gut, home to surgical wounds, and cause infection (Trojan Horse Hypothesis). METHODS: MRSA gut colonization was achieved by disrupting the microbiota with antibiotics, imposing a period of starvation and introducing MRSA via gavage. Next, mice were subjected to a surgical injury (30% hepatectomy) and rectus muscle injury and ischemia before skin closure. All wounds were cultured before skin closure. To control for postoperative wound contamination, reiterative experiments were performed in mice in which the closed wound was painted with live MRSA for 2 consecutive postoperative days. To rule out extracellular bacteremia as a cause of wound infection, MRSA was injected intravenously in mice subjected to rectus muscle ischemia and injury. RESULTS: All wound cultures were negative before skin closure, ruling out intraoperative contamination. Out of 40 mice, 4 (10%) developed visible abscesses. Nine mice (22.5%) had MRSA positive cultures of the rectus muscle without visible abscesses. No SSIs were observed in mice injected intravenously with MRSA. Wounds painted with MRSA after closure did not develop infections. Circulating neutrophils from mice captured by flow cytometry demonstrated MRSA in their cytoplasm. CONCLUSIONS: Immune cells as Trojan horses carrying gut-derived MRSA may be a plausible mechanism of SSIs in the absence of direct contamination.

Metabolic Mitigation of Staphylococcus aureus Vancomycin Intermediate-Level Susceptibility.

Staphylococcus aureus is a major human pathogen whose infections are increasingly difficult to treat due to increased antibiotic resistance, including resistance to vancomycin. Vancomycin-intermediate S. aureus (VISA) strains develop resistance to vancomycin through adaptive changes that are incompletely understood. Central to this adaptation are metabolic changes that permit growth in the presence of vancomycin. To define the metabolic changes associated with adaptive resistance to vancomycin in S. aureus, the metabolomes of a vancomycin-sensitive and VISA strain pair isolated from the same patient shortly after vancomycin therapy began and following vancomycin treatment failure were analyzed. The metabolic adaptations included increases in acetogenesis, carbon flow through the pentose phosphate pathway, wall teichoic acid and peptidoglycan precursor biosynthesis, purine biosynthesis, and decreased tricarboxylic acid (TCA) cycle activity. The significance of these metabolic pathways for vancomycin-intermediate susceptibility was determined by assessing the synergistic potential of human-use-approved inhibitors of these pathways in combination with vancomycin against VISA strains. Importantly, inhibitors of amino sugar and purine biosynthesis acted synergistically with vancomycin to kill a diverse set of VISA strains, suggesting that combinatorial therapy could augment the efficacy of vancomycin even in patients infected with VISA strains.

Treatment of Staphylococcus aureus Infections.

Staphylococcus aureus, although generally identified as a commensal, is also a common cause of human bacterial infections, including of the skin and other soft tissues, bones, bloodstream, and respiratory tract. The history of S. aureus treatment is marked by the development of resistance to each new class of antistaphylococcal antimicrobial drugs, including the penicillins, sulfonamides, tetracyclines, glycopeptides, and others, complicating therapy. S. aureus isolates identified in the 1960s were sometimes resistant to methicillin, a ß-lactam antimicrobial active initially against a majority S. aureus strains. These MRSA isolates, resistant to nearly all ß-lactam antimicrobials, were first largely confined to the health care environment and the patients who attended it. However, in the mid-1990s, new strains, known as community-associated (CA-) MRSA strains, emerged. CA-MRSA organisms, compared with health care-associated (HA-) MRSA strain types, are more often susceptible to multiple classes of non ß-lactam antimicrobials. While infections caused by methicillin-susceptible S. aureus (MSSA) strains are usually treated with drugs in the ß-lactam class, such as cephalosporins, oxacillin or nafcillin, MRSA infections are treated with drugs in other antimicrobial classes. The glycopeptide drug vancomycin, and in some countries teicoplanin, is the most common drug used to treat severe MRSA infections. There are now other classes of antimicrobials available to treat staphylococcal infections, including several that have been approved after 2009. The antimicrobial management of invasive and noninvasive S. aureus infections in the ambulatory and in-patient settings is the topic of this review. Also discussed are common adverse effects of antistaphylococcal antimicrobial agents, advantages of one agent over another for specific clinical syndromes, and the use of adjunctive therapies such as surgery and intravenous immunoglobulin. We have detailed considerations in the therapy of noninvasive and invasive S. aureus infections. This is followed by sections on specific clinical infectious syndromes including skin and soft tissue infections, bacteremia, endocarditis and intravascular infections, pneumonia, osteomyelitis and vertebral discitis, epidural abscess, septic arthritis, pyomyositis, mastitis, necrotizing fasciitis, orbital infections, endophthalmitis, parotitis, staphylococcal toxinoses, urogenital infections, and central nervous system infections.

A Placebo-Controlled Trial of Antibiotics for Smaller Skin Abscesses.

BACKGROUND: Uncomplicated skin abscesses are common, yet the appropriate management of the condition in the era of community-associated methicillin-resistant Staphylococcus aureus (MRSA) is unclear. METHODS: We conducted a multicenter, prospective, double-blind trial involving outpatient adults and children. Patients were stratified according to the presence of a surgically drainable abscess, abscess size, the number of sites of skin infection, and the presence of nonpurulent cellulitis. Participants with a skin abscess 5 cm or smaller in diameter were enrolled. After abscess incision and drainage, participants were randomly assigned to receive clindamycin, trimethoprim-sulfamethoxazole (TMP-SMX), or placebo for 10 days. The primary outcome was clinical cure 7 to 10 days after the end of treatment. RESULTS: We enrolled 786 participants: 505 (64.2%) were adults and 281 (35.8%) were children. A total of 448 (57.0%) of the participants were male. S. aureus was isolated from 527 participants (67.0%), and MRSA was isolated from 388 (49.4%). Ten days after therapy in the intention-to-treat population, the cure rate among participants in the clindamycin group was similar to that in the TMP-SMX group (221 of 266 participants [83.1%] and 215 of 263 participants [81.7%], respectively; P=0.73), and the cure rate in each active-treatment group was higher than that in the placebo group (177 of 257 participants [68.9%], P<0.001 for both comparisons). The results in the population of patients who could be evaluated were similar. This beneficial effect was restricted to participants with S. aureus infection. Among the participants who were initially cured, new infections at 1 month of follow-up were less common in the clindamycin group (15 of 221, 6.8%) than in the TMP-SMX group (29 of 215 [13.5%], P=0.03) or the placebo group (22 of 177 [12.4%], P=0.06). Adverse events were more frequent with clindamycin (58 of 265 [21.9%]) than with TMP-SMX (29 of 261 [11.1%]) or placebo (32 of 255 [12.5%]); all adverse events resolved without sequelae. One participant who received TMP-SMX had a hypersensitivity reaction. CONCLUSIONS: As compared with incision and drainage alone, clindamycin or TMP-SMX in conjunction with incision and drainage improves short-term outcomes in patients who have a simple abscess. This benefit must be weighed against the known side-effect profile of these antimicrobials. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT00730028 .).

Evaluation of serotypes 5 and 8 capsular polysaccharides in protection against Staphylococcus aureus in murine models of infection.

Staphylococcus aureus is the leading cause of nosocomial and community-acquired infections, including soft tissue and skin infections and bacteremia. However, efforts to develop an effective vaccine against S. aureus infections have not been successful. We evaluated serotypes 5 and 8 capsule polysaccharides (CP) CRM197 conjugates as vaccine candidates in murine models of bacteremia, lethal sepsis, and skin infection. The conjugate vaccines elicited a good antibody response, and active immunization of CP5-CRM or CP8-CRM conjugates protected against staphylococcal bacteremia. In the skin infection model, CP8-CRM but not CP5-CRM protected against dermonecrosis, and CP8-CRM immunization significantly decreased the bacterial burden in the lesion. However, neither CP5-CRM nor CP8-CRM protected against mortality in the lethal sepsis model. The results indicate the capsular vaccines elicit protection against some, but not all, aspects of staphylococcal infection.

Daptomycin for Complicated Skin Infections: A Randomized Trial.

BACKGROUND: Complicated skin and skin structure infections (cSSSI) are common in children. Due to safety and resistance issues with recommended agents, new treatment options would be advantageous. METHODS: Multicenter, evaluator-blinded clinical trial. Patients 1 to 17 years old with cSSSI caused by Gram-positive pathogens were randomized 2:1 to intravenous daptomycin or standard-of-care (SOC) treatment for ≤14 days. Daptomycin was administered once daily with dosing by patient age: 12 to 17 years, 5 mg/kg; 7 to 11 years, 7 mg/kg; 2 to 6 years, 9 mg/kg; 12 to 23 months, 10 mg/kg. The primary objective was to evaluate daptomycin safety. The secondary objective was to assess the efficacy of daptomycin compared with SOC. The intent-to-treat (ITT) population consisted of all randomized patients with any dose of study drug. RESULTS: The ITT population comprised 257 daptomycin and 132 SOC patients (primarily clindamycin or vancomycin); 35% had confirmed methicillin-resistant Staphylococcus aureus. The most common adverse events were diarrhea (7% daptomycin, 5% SOC) and increased creatine phosphokinase (6% daptomycin, 5% SOC). The proportions of safety population patients with treatment-related adverse events were similar between the daptomycin (14%) and SOC (17%) groups. Clinical success rates (blinded evaluator-assessed complete/partial resolution of cSSSI signs and symptoms 7-14 days after end-of-treatment) in the ITT population were also similar for the daptomycin (91%) and SOC groups. CONCLUSIONS: Once-daily daptomycin was well tolerated, with safety and efficacy comparable to SOC in children/adolescents with cSSSI caused by Gram-positive pathogens, including community-acquired methicillin-resistant S aureus.

Identification of source and sink populations for the emergence and global spread of the East-Asia clone of community-associated MRSA.

BACKGROUND: Our understanding of the factors influencing the emergence, dissemination and global distribution of epidemic clones of bacteria is limited. ST59 is a major epidemic clone of community-associated MRSA in East Asia, responsible for extensive morbidity and mortality, but has a much lower prevalence in other parts of the world. The geographic origin of ST59 and its international routes of dissemination are unclear and disputed in the literature. RESULTS: To investigate the origin and spread of the ST59 clone, we obtained whole genome sequences of isolates from four continents, sampled over more than a decade, and carried out a time-scaled phylogeographic analysis. We discover that two distinct ST59 clades emerged concurrently, in East Asia and the USA, but underwent clonal expansion at different times. The East Asia clade was strongly enriched for gene determinants associated with antibiotic resistance, consistent with regional differences in antibiotic usage. Both clones spread independently to Australia and Europe, and we found evidence of the persistence of multi-drug resistance following export from East Asia. Direct transfer of strains between Taiwan and the USA was not observed in either direction, consistent with geographic niche exclusion. CONCLUSIONS: Our results resolve a longstanding controversy regarding the origin of the ST59 clone, revealing the major global source and sink populations and routes for the spread of multi-drug resistant clones. Additionally, our findings indicate that diversification of the accessory genome of epidemic clones partly reflects region-specific patterns of antibiotic usage, which may influence bacterial fitness after transmission to different geographic locations.

Impact of Staphylococcus aureus USA300 Colonization and Skin Infections on Systemic Immune Responses in Humans.

Staphylococcus aureus is both a commensal and a pathogen, and USA300, a strain that is usually methicillin-resistant but can sometimes be methicillin-susceptible, has been causing skin and soft tissue infections (SSTIs) in epidemic proportions among otherwise healthy individuals. Although many people are colonized with S. aureus strains, including some with USA300, few of these colonized individuals develop SSTIs. This prompts the hypothesis that infections may develop in individuals with somewhat reduced innate and/or adaptive immune responses to S. aureus, either because prior S. aureus colonization has dampened such responses selectively, or because of more globally reduced immune reactivity. In this study, we analyzed the S. aureus colonization status and PBMC responses to innate and adaptive stimuli in 72 patients with SSTIs and 143 uninfected demographically matched controls. Contrary to the hypothesis formulated, PBMCs from infected patients obtained at the time of infection displayed enhanced innate cytokine production upon restimulation compared with PBMCs from controls, a difference that disappeared after infection resolution. Notably, PBMCs from patients infected with a documented USA300 SSTI displayed greater innate cytokine production than did those from patients infected with documented non-USA300 genotypes. Moreover, colonization with USA300 in infected patients, regardless of their infecting strain, correlated with increased production of IL-10, IL-17A, and IL-22 compared with patients colonized with non-USA300 subtypes. Thus, our results demonstrate that infected patients associated with USA300 either as an infecting strain, or as a colonizing strain, have systemic immune responses of greater magnitude than do those associated with other S. aureus subtypes.

Intrahost Evolution of Methicillin-Resistant Staphylococcus aureus USA300 Among Individuals With Reoccurring Skin and Soft-Tissue Infections.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) USA300 is the leading cause of MRSA infections in the United States and has caused an epidemic of skin and soft-tissue infections. Recurrent infections with USA300 MRSA are common, yet intrahost evolution during persistence on an individual has not been studied. This gap hinders the ability to clinically manage recurrent infections and reconstruct transmission networks. METHODS: To characterize bacterial intrahost evolution, we examined the clinical courses of 4 subjects with 3-6 recurrent USA300 MRSA infections, using patient clinical data, including antibiotic exposure history, and whole-genome sequencing and phylogenetic analysis of all available MRSA isolates (n = 29). RESULTS: Among sequential isolates, we found variability in diversity, accumulation of mutations, and mobile genetic elements. Selection for antimicrobial-resistant populations was observed through both an increase in the number of plasmids conferring multidrug resistance and strain replacement by a resistant population. Two of 4 subjects had strain replacement with a genetically distinct USA300 MRSA population. DISCUSSIONS: During a 5-year period in 4 subjects, we identified development of antimicrobial resistance, intrahost evolution, and strain replacement among isolates from patients with recurrent MRSA infections. This calls into question the efficacy of decolonization to prevent recurrent infections and highlights the adaptive potential of USA300 and the need for effective sampling.

Decreasing Incidence of Skin and Soft Tissue Infections With a Seasonal Pattern at an Academic Medical Center, 2006-2014.

Background. The incidence of skin and soft tissue infections (SSTIs) in the United States increased sharply after 2000 with the emergence of USA300 methicillin-resistant Staphylococcus aureus. We examined trends in SSTI incidence in 2006-2014 at the University of Chicago Medicine (UCM). Methods. Data were obtained for patient encounters at UCM with an International Classification of Diseases, Ninth Revision-coded SSTI diagnosis between January 1, 2006 and March 31, 2014. Incidence density was calculated per 1000 encounters by quarter and year. Encounters were stratified by inpatient, outpatient clinic and emergency department (ED) encounters and by age group, gender, and race. Poisson regression was used to assess change over time. Results. In 2006-2014, data were collected for 38 201 SSTI-associated encounters among 31 869 subjects. Among all patients treated at UCM, there was a decrease of 1% per year in the incidence of SSTIs during 2006-2013, with an overall decrease of 16%. There was a significant decrease in SSTI-related encounters among inpatients (rate ratio [RR] = 0.97; 95% confidence interval [CI], .96-.98), ED patients (RR = 0.98; 95% CI, .97-.98), adults (RR = 0.98; 95% CI, .97-.98), children (RR = 0.96; 95% CI, .95-.97), and African Americans (RR = 0.99; 95% CI, .98-.99). There was an annual seasonal trend, with the peak incidence occurring during the late summer. Conclusions. The incidence of SSTIs at UCM decreased in children and adults with seasonal variation, peaking during the summer months. This suggests a reversal of the massive increase in SSTI incidence in the United States after 2000.

Proteomic Identification of saeRS-Dependent Targets Critical for Protective Humoral Immunity against Staphylococcus aureus Skin Infection.

Recurrent Staphylococcus aureus skin and soft tissue infections (SSTIs) are common despite detectable antibody responses, leading to the belief that the immune response elicited by these infections is not protective. We recently reported that S. aureus USA300 SSTI elicits antibodies that protect against recurrent SSTI in BALB/c but not C57BL/6 mice, and in this study, we aimed to uncover the specificity of the protective antibodies. Using a proteomic approach, we found that S. aureus SSTI elicited broad polyclonal antibody responses in both BALB/c and C57BL/6 mice and identified 10 S. aureus antigens against which antibody levels were significantly higher in immune BALB/c serum. Four of the 10 antigens identified are regulated by the saeRS operon, suggesting a dominant role for saeRS in protection. Indeed, infection with USA300Δsae failed to protect against secondary SSTI with USA300, despite eliciting a strong polyclonal antibody response against antigens whose expression is not regulated by saeRS. Moreover, the antibody repertoire after infection with USA300Δsae lacked antibodies specific for 10 saeRS-regulated antigens, suggesting that all or a subset of these antigens are necessary to elicit protective immunity. Infection with USA300Δhla elicited modest protection against secondary SSTI, and complementation of USA300Δsae with hla restored protection but incompletely. Together, these findings support a role for both Hla and other saeRS-regulated antigens in eliciting protection and suggest that host differences in immune responses to saeRS-regulated antigens may determine whether S. aureus infection elicits protective or nonprotective immunity against recurrent infection.

Pediatric Staphylococcus aureus Isolate Genotypes and Infections from the Dawn of the Community-Associated Methicillin-Resistant S. aureus Epidemic Era in Chicago, 1994 to 1997.

Widespread infections with community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) have occurred in the United States with the dissemination of the USA300 strain beginning in 2000. We examined 105 isolates obtained from children treated at the University of Chicago from 1994 to 1997 (75 methicillin-susceptible S. aureus [MSSA] and 30 MRSA isolates) in order to investigate for possible evidence of USA300 during this period. Infections were defined epidemiologically based on medical record review. The isolates underwent multilocus sequence typing (MLST), as well as assays for the Panton-Valentine leukocidin (PVL) genes, the protein A gene (spa), and arcA and opp3, proxy markers for the arginine catabolic mobile element (ACME), characteristic of USA300 MRSA. MRSA isolates also underwent staphylococcal cassette chromosome mec (SCCmec) typing and pulsed-field gel electrophoresis (PFGE) subtyping. MSSA isolates belonged to 17 sequence type (ST) groups. The 12 epidemiologically defined CA-MRSA infection isolates were either ST1 (n = 4) or ST8 (n = 8). They belonged to 3 different PFGE types: USA100 (n = 1), USA400 (n = 5), and USA500 (n = 6). Among the CA-MRSA infection isolates, 8 (67%) were PVL(+). None of the MRSA or MSSA isolates contained arcA or opp3. Only one MRSA isolate was USA300 by PFGE. This was a health care-associated (HA) MRSA isolate, negative for PVL, that carried SCCmec type II. USA300 with its characteristic features was not identified in the collection from the years 1994 to 1997.

Staphylococcus aureus metabolic adaptations during the transition from a daptomycin susceptibility phenotype to a daptomycin nonsusceptibility phenotype.

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. The success of S. aureus as a pathogen is due in part to its many virulence determinants and resistance to antimicrobials. In particular, methicillin-resistant S. aureus has emerged as a major cause of infections and led to increased use of the antibiotics vancomycin and daptomycin, which has increased the isolation of vancomycin-intermediate S. aureus and daptomycin-nonsusceptible S. aureus strains. The most common mechanism by which S. aureus acquires intermediate resistance to antibiotics is by adapting its physiology and metabolism to permit growth in the presence of these antibiotics, a process known as adaptive resistance. To better understand the physiological and metabolic changes associated with adaptive resistance, six daptomycin-susceptible and -nonsusceptible isogenic strain pairs were examined for changes in growth, competitive fitness, and metabolic alterations. Interestingly, daptomycin nonsusceptibility coincides with a slightly delayed transition to the postexponential growth phase and alterations in metabolism. Specifically, daptomycin-nonsusceptible strains have decreased tricarboxylic acid cycle activity, which correlates with increased synthesis of pyrimidines and purines and increased carbon flow to pathways associated with wall teichoic acid and peptidoglycan biosynthesis. Importantly, these data provided an opportunity to alter the daptomycin nonsusceptibility phenotype by manipulating bacterial metabolism, a first step in developing compounds that target metabolic pathways that can be used in combination with daptomycin to reduce treatment failures.