Persistent Bacteremia Due to Methicillin-Resistant Staphylococcus aureus Infection Is Associated with agr Dysfunction and Low-Level In Vitro Resistance to Thrombin-Induced Platelet Microbicidal Protein

Journal of Infectious Diseases, Sep 2004

Background. The causes of persistent bacteremia (PB) due to methicillin-resistant Staphylococcus aureus (MRSA) are poorly understood. This investigation examined potential associations between PB with key clinical features and several in vitro bacterial genotypic and phenotypic characteristics, in isolates from 1 institution. Methods. Pulsed-field gel electrophoresis (PFGE) relatedness, thrombin-induced platelet microbicidal protein (tPMP)-susceptibility phenotype, accessory gene regulator (agr) genotype and functionality (via δ-lysin production), and autolysis phenotypes were assessed in MRSA isolates from the bloodstream of 21 prospectively identified patients with PB (blood cultures positive after ⩾7 days of therapy) and of 18 patients with resolving bacteremia (RB) (sterile blood cultures within the first 2–4 days of therapy) due to MRSA. Results. The 2 groups had comparable baseline characteristics but differed in their clinical courses (e.g., endocarditis was more frequent in patients with PB than in those with RB [43% vs. 0%, respectively; P = .0016]); isolates from patients with PB exhibited higher rates of (1) survival in vitro after exposure to tPMP (22.4 ± 14.8% vs. 11.6 ± 6.5%, respectively; P = .005); (2) defective δ-lysin production (71.4% vs. 38.9%, respectively; P = .057); (3) non-agr genotype II profile (100% vs. 77.8%, respectively; P = .037); and (4) overrepresentation of a specific PFGE genotype (85.7% vs. 44.4%, respectively; P = .015). Conclusions. Isolates from patients with PB differed from those in patients with RB, in several in vitro characteristics. Further studies will be necessary to define how these factors might affect clinical outcome.

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Persistent Bacteremia Due to Methicillin-Resistant Staphylococcus aureus Infection Is Associated with agr Dysfunction and Low-Level In Vitro Resistance to Thrombin-Induced Platelet Microbicidal Protein

JID Persistent Bacteremia Due to Methicillin-Resistant Staphylococcus aureus Infection Is Associated with agr Dysfunction and Low-Level In Vitro Resistance to Thrombin-Induced Platelet Microbicidal Protein Vance G. Fowler 0 1 2 Jr. 0 1 2 George Sakoulas 0 1 2 6 Lauren M. McIntyre 0 1 2 5 Venkata G. Meka 0 1 2 4 Robert D. Arbeit 0 1 2 9 Christopher H. Cabell 0 1 2 Martin E. Stryjewski 0 1 2 George M. Eliopoulos 0 1 2 4 L. Barth Reller 0 1 2 7 G. Ralph Corey 0 1 2 Tiffanny Jones 0 1 2 8 Natalie Lucindo 0 1 2 8 Michael R. Yeaman 0 1 2 3 8 Arnold S. Bayer 0 1 2 3 8 0 be related to specific features of the infecting organisms 1 This observation suggests that such complications may 2 Department of Medicine, Divisions of 3 Geffen School of Medicine, UCLA , Los Angeles, California 4 Beth Israel Deaconess Medical Center and Harvard Medical School 5 Computational Genomics, Purdue University , West Lafayette, Indiana 6 Crystal Run Healthcare , Middletown , New York 7 Clinical Microbiology Laboratory, Duke University Medical Center , Durham, North Carolina 8 St. John's Cardiovascular Research Center, Research and Education Institute, and Division of Infectious Diseases, Harbor-UCLA Medical Center , Torrance 9 Paratek Pharmaceuticals , Boston, Massachusetts Background. The causes of persistent bacteremia (PB) due to methicillin-resistant Staphylococcus aureus (MRSA) are poorly understood. This investigation examined potential associations between PB with key clinical features and several in vitro bacterial genotypic and phenotypic characteristics, in isolates from 1 institution. Methods. Pulsed-field gel electrophoresis (PFGE) relatedness, thrombin-induced platelet microbicidal protein (tPMP)-susceptibility phenotype, accessory gene regulator (agr) genotype and functionality (via d-lysin production), and autolysis phenotypes were assessed in MRSA isolates from the bloodstream of 21 prospectively identified patients with PB (blood cultures positive after 7 days of therapy) and of 18 patients with resolving bacteremia (RB) (sterile blood cultures within the first 2-4 days of therapy) due to MRSA. Results. The 2 groups had comparable baseline characteristics but differed in their clinical courses (e.g., endocarditis was more frequent in patients with PB than in those with RB [43% vs. 0%, respectively; P p .0016]); isolates from patients with PB exhibited higher rates of (1) survival in vitro after exposure to tPMP (22.4 14.8% vs. 11.6 6.5%, respectively; P p .005); (2) defective d-lysin production (71.4% vs. 38.9%, respectively; P p .057); (3) non-agr genotype II profile (100% vs. 77.8%, respectively; P p .037); and (4) overrepresentation of a specific PFGE genotype (85.7% vs. 44.4%, respectively; P p .015). Conclusions. Isolates from patients with PB differed from those in patients with RB, in several in vitro characteristics. Further studies will be necessary to define how these factors might affect clinical outcome. Although bacteremia due to Staphylococcus aureus is common, complications of this condition (e.g., endocarditis and persistent bacteremia [PB]) are infrequent. - those associated with b-lactam therapy for methicillin-susceptible staphylococcemic syndromes [3]. A number of recent studies have strongly suggested that platelets play a key role in host defense against endovascular infections, including those due to S. aureus. This host-defense property of platelets has been closely linked to their secretion of platelet microbicidal proteins (PMPs) in response to agonists (e.g., thrombin) generated at sites of endovascular infections [4]. PMPs can kill organisms, including S. aureus, that commonly enter the bloodstream [4–15]. Compared with isolates from other clinical sources, those associated with human endovascular infections more frequently exhibit stable, low-level resistance to the microbicidal activities of PMPs in vitro [6, 8, 9]. In experimental infective endocarditis (IE) due to S. aureus, infection due to relatively PMP-resistant strains is associated with larger endocardial vegetations, higher bacterial densities in vegetations and renal abscesses, and longer durations of bacteremia, compared with infection caused by isogenic, PMPsusceptible strains [11]. These studies have suggested that, in S. aureus, resistance to PMP may either directly contribute to PB or serve as a surrogate marker for such outcomes. The accessory gene regulator (agr) locus of S. aureus is a pivotal global regulon that coordinates the expression of a number of virulence genes in this organism, including those for hemolysins and autolysins [16, 17]. S. aureus strains have been grouped on the basis of secretion of specific autoinducing oligopeptides (AIPs I– IV) that govern and autoregulate the quorum-sensing response of this organism [18, 19]. Jarraud et al. recently reported an overrepresentation of agr AIP genotype II in S. aureus isolates from patients with IE [18]. Moreover, we and others have recently shown, in laboratory-derived S. aureus, an interesting association between (1) exposures to subinhibitory levels of vancomycin, (2) loss of agr function, (3) intermediate-level glycopeptide resistance (GISA), (4) decreased autolysis, and (5) reduced susceptibility to PMPs [20–22]; however, there has been no comprehensive study of these factors and their potential relationship to the development of PB in isolates from patients with well-defined patterns of S. aureus infection. During the past decade, we have prospectively collected, at a single large referral center, both S. aureus isolates from the bloodstream and detailed clinical information from all patients with S. aureus bacteremia [23]. Using this database, we have identified all MRSA isolates associated with PB and have analyzed their phenotypic and genotypic characteristics, including their in vitro PMP-susceptibility profiles, agr AIP genotype, autolytic profiles, and d-lysin production (as a marker of agr function) [22]. PATIENTS, MATERIALS, AND METHODS Clinical characteristics of patients. Research guidelines of Duke University and of the University of California Los Angeles (UCLA) were followed in the conduct of the clinical research, and the study was approved by the Duke University Medical Center (DUMC) Institutional Review Board and the Compliance Office at the Research & Education Institute, HarborUCLA. As reported elsewhere [10], consecutive S. aureus isolates from the bloodstream and corresponding clinical data have been prospectively identified and collected since September 1994 from all adult patients hospitalized at DUMC who had bacteremia due solely to S. aureus bacteremia. In brief, daily reports from the microbiology laboratory were received for all patients at DUMC who had clinical evidence of infection and at least 1 blood-culture isolate positive for S. aureus. Clinical data were collected at the time of hospitalization, by investigators well versed in clinical infectious diseases and were entered into a computer database (ACCESS; Microsoft). The APACHE II score [24] was calculated for each patient, on the calendar date of the initial blood culture that showed infection by S. aureus. Patients were excluded from the current study if they were outpatients, were !18 years of age, had polymicrobial infection, were neutropenic (absolute neutrophil count, !1.0 109 cells/ L), or died before being evaluated by the investigators. Corticosteroid use was categorized as either “chronic, low-dose” therapy or “acute, high-dose” therapy [24]. Isolation and characterization of S. aureus isolates. Blood-culture isolates were identified as S. aureus and were stored for future use, as described elsewhere [25]. For the present study, only the initial S. aureus–positive culture at DUMC was analyzed by the clinical microbiology laboratory. In brief, isolates were identified as S. aureus by hemolysis on sheepblood agar, by Gram staining, and by Staphaurex latex-agglutination assay (Murex Diagnostics). Susceptibilty to methicillin was determined by the oxacillin disk test performed as specified by the NCCLS [26]. Definitions. The decision to obtain initial and follow-up blood-culture isolates was made by the medical team caring for the patient. For this study, 2 groups of patients were differentiated on the basis of the microbiologic pattern of their S. aureus bacteremia: PB was defined as documented MRSA bacteremia of 7 days duration while the patient was receiving antimicrobial therapy to which the isolate was susceptible; resolving bacteremia (RB) was defined as documented MRSA bacteremia in the initial positive blood culture, with all subsequent blood cultures being negative. Patients with RB had to have at least 1 set of follow-up blood cultures 2–4 days after the initial positive blood culture. Septic shock [27], a soft-tissue source [28] or an intravascular-catheter source [29], and the presence of IE [30] were defined according to widely accepted criteria. Aminoglycoside use and rifampin use were defined as administration of these agents at any time during the 14 days after the index blood culture. Determination of d-lysin activity. d-Lysin is encoded after activation of the agr operon [17]; consequently, dysfunction or deletion of agr results in the absence of d-lysin production. Agar-plate assays for d-lysin were performed as described elsewhere [22], by 2 investigators (G.S. and V.M.) who were located at a center separate from that where the isolates were obtained and who were blinded as to any clinical information or any patient identifiers. Identification of agr type. Because earlier studies have documented an association between dysfunction of agr AIP genotype II, defective autolysis, and MRSA-GISA phenotypes [20], we focused on whether the strains studied in the present investigation were agr AIP genotype II or non–agr AIP genotype II [20]. MRSA isolates from the bloodstream were studied by PCR using agr AIP genotype II–specific primers, as reported elsewhere [31]. The 5 primer sequence (bp 155–163; GenBank accession number AF001782) was 5 -GTAGAGCCGTATTGATTCC-3 ; the 3 primer sequence (bp 600–618; GenBank accession number AF001782) was 5 -GTATTTTCATCTCTTTAAGG-3 . The reaction conditions were as described elsewhere [21]. PCR products were analyzed by standard methods of agarose-gel electrophoresis. A8832, an agr AIP genotype II strain, was used as a positive control [17, 32]. Determination of lysis profiles. Triton-induced lysis is an established surrogate for the measurement of the propensity for autolysis in S. aureus strains [20]. Overnight cultures were diluted 1:1000 in brain-heart–infusion broth and were grown to an OD630 of 0.6–0.8. Cells were aliquoted to a clean sterile microcentrifuge tube, were cooled on ice, and were spun for 30 s in a table-top microcentrifuge. Bacterial pellets were washed twice with cold distilled water and were resuspended to an OD630 of ∼1.0, in buffer comprising 0.01% Triton X-100, 50 mmol glycine/L, pH 8.0. The suspension (200 mL) was aliquoted, in duplicate, onto a sterile 96-well flat-bottomed plastic tissue-culture plate (Costar) and incubated at 35 C, with gentle agitation. OD630 was measured, spectrophotometrically, at baseline (time 0 h), at hourly intervals for 6 h, and at 16 h and 24 h. Percentage of lysis at each time point was defined as the percentage decrease in OD630, compared with the baseline reading. On the basis of earlier studies [13], “defective autolysis” was defined as an OD630 decrease of !30% after 5 h of exposure to Triton, compared with the baseline reading. In vitro assay for susceptibility to PMP. PMP-susceptibility assays in vitro were performed by investigators (T.J., A.S.B., and M.R.Y.) who were blinded to any clinical details. Bacterial susceptibility to thrombin-induced PMP (tPMP) (secreted by mammalian platelets stimulated by thrombin) was assayed in vitro, as described elsewhere [33]; the results are expressed as the percentage of the initial inoculum (103 cfu) that survived exposure to tPMP at a specific activity of 2 mg/ mL. For each isolate, susceptibility to tPMP was expressed as Table 1. Baseline characteristics of 39 patients with either resolving or persistent methicillin-resistant Staphylococcus aureus bacteremia. Resolving bacteremia (n p 18) Persistent bacteremia (n p 21) NOTE. Data are either no. (%) of patients in subgroup or mean unless other indicated. DUMC, Duke University Medical Center. the average of the results of 2 independent experimental assays, performed on separate days. Preparation of tPMP, quality control of peptide activity, and the quantitative in vitro–assay methods for tPMP-susceptibility phenotypes have been described in detail elsewhere [12]. Pulsed-field gel electrophoresis (PFGE). Two sets of PFGE analyses were performed. The first analysis was to determine the relationships among the chromosomal genotypes of the initial MRSA blood-culture isolates from patients with RB and patients with PB [34]. The second analysis was to confirm that PB represented ongoing infection with a single strain, rather than reinfection with a different strain; for the latter analysis, the initial isolate from the bloodstream of each patient was compared with a second isolate, cultured 7 days later, from the bloodstream of the same patient. For PFGE, DNA was prepared as described elsewhere [35], with the following modifications: 5 mL of a solution of 1 mg of lysostaphin/mL and 20 mmol sodium acetate/mL (pH 4.5) was used to lyse cells, and 40 U of SmaI was used to digest recovered DNA. Digest fragments were separated by use of a CHEF-DR II Pulsed Field Electrophoresis System (Bio-Rad Laboratories) with a ramped pulse of 15–55 s at 200 V for 22 h at 14 C. Gels were stained in a 0.1% ethidium bromide solution for 15 min and were destained in distilled water for 2 5 Blood samples cultured Antibiotic 38.0 C for 72 h Resolving bacteremia (n p 18) Persistent bacteremia (n p 21) Note. Data are no. (%) of patients in subgroup, unless otherwise indicated. h. Stained gels were photographed by a Polaroid MP-4 Land Camera (Polaroid), and the photographs were digitized. Statistical methods. Categorical variables, including demographic, clinical, and microbiologic characteristics (e.g., sex of patient and bacterial genotype), were analyzed, and the PB group and the RB group were compared by a x2 test for association; P values were calculated exactly. Continuous variables (e.g., APACHE II) were analyzed by Student’s t test. Interactions between demographic, clinical, and microbiologic characteristics were evaluated in multivariate logistic models (SAS version 8.1). Genotypic classification of the isolates was determined on the basis of the PFGE pattern [34]. The digitized PFGE profiles were analyzed by the BioNumerics program (Applied Maths). PFGE genotypes were defined as having 0.85 relatedness. Dice coefficients (pairwise similarity) were calculated for each pair of isolates; and a dendrogram was constructed by the unweighted pair-group method of analysis [36], with an optimization value of 0.50% and a position tolerance of 1.25%– 1.35% (end of the fingerprint). Between 2 February 1995 and 26 November 2000, a total of 337 patients with MRSA bacteremia were prospectively identified. Of these 337 patients, 21 (6.2%) met the criteria for PB and 107 (31.8%) met the criteria for RB; 18 of the latter group were randomly selected for inclusion in the present study. The MIC of vancomycin in isolates from the the bloodstream of these 39 patients included in the study was 0.25–1.0 mg/mL and did not differ between the PB group and the RB group. No GISA strains were observed. At the time when the initial MRSA blood-culture isolate was extracted, both the PB group and the RB group had similar demographic, clinical, and laboratory characteristics (table 1). Overall, the patients tended to be men (64.1%), white (56.4%), and 165 years of age (61%). Approximately half the infections in each group were hospital acquired, with an intravascular device being the most common presumed source of bacteremia. All of the patients with community-acquired infection had experienced a recent and relatively intensive contact with healthcare services (e.g., hospitalization during the previous 3 months or dialysis treatment); of the 39 patients included in the present study, 13 (33%; 8 with PB, 5 with RB) were transferred to DUMC from another medical facility. Nearly all patients received vancomycin as their principal anti-MRSA therapy. The average interval between the initial MRSA-positive blood-culture isolate and the initiation of vancomycin therapy was similar for the RB group and the PB group (0.5 1.3 days and 0.4 1.1 days, respectively; P p .75); likewise, the average time between the initial MRSA-positive blood-culture isolate and the removal of an infected intravascular-device source was not significantly different between the RB group and the PB group (1.1 2.5 days and 4.9 6.3 days, respectively; P p .1). In contrast, the 2 groups differed significantly in characteristics associated with clinical course and outcome (table 2). Patients with PB were febrile longer, received more-extensive blood-culture monitoring, received longer courses of antibiotics, and were more likely to have received concomitant ami Figure 1. (Continued.) noglycosides. In addition, compared with patients with RB, patients with PB tended to have higher trough vancomycin levels, although this difference was not statistically significant. Moreover, patients with PB were significantly more likely to have a diagnosis of IE than were patients with RB (43% vs. 0%, respectively; P p .002). Also, patients with PB were significantly more likely to have undergone both transthoracic (90% vs. 39%, respectively; P p .0015) and transesophageal (81% vs. 44%, respectively; P p .024) echocardiography. Death from all causes was more common in patients with PB (43%) than in patients with RB (17%), although the difference was not statistically significant. Genotypic characterization of isolates. The PFGE analyses revealed a moderate diversity of genotypes in the 39 initial blood-culture isolates; the resulting dendrogram had a minimum overall relatedness of 0.55 (figure 1A). The most prevalent PFGE genotype (designated genotype “A”) comprised 2 closely related genotypes [34], designated “A1” (23 isolates) and “A2” (3 isolates). PFGE genotype B also comprised 2 closely related genotypes, designated “B1” (6 isolates) and “B2” (1 isolate). The remaining 6 isolates each represented a distinctly different genotype [34]. Isolates from patients with PB were significantly more likely to be PFGE genotype A than were isolates from patients with RB (18/21 [86%] vs. 8/18 [44%], respectively; P p .02) (figure 1A and table 3). Paired isolates from the initial MRSA-positive blood culture and from a blood culture 7 days later were available for 20 of the 21 patients with PB. In all 20 of these patients, the paired isolates had indistinguishable genotypes, a finding consistent with the presence of a single persistent infection (figure 1B). In vitro tPMP-susceptibility phenotype. Compared with isolates from patients with RB, those from patients with PB exhibited a significantly higher percentage of survival after in vitro exposure to low levels of tPMP-1 (table 3); of interest is that the percentage of survival was significantly higher for the 9 isolates from the bloodstream of patients with IE than it was for the remaining 30 isolates (28.1% 15.4% vs. 14.2% 10.2%, respectively; P p .03). agr genotype and d-lysin activity. Of the 39 tested isolates, 4 were agr AIP genotype II; all 4 of these isolates were from patients with RB, and 3 of them were PFGE genotype non-A (table 3). The mean percentage of survival after exposure to tPMP in vitro was significantly lower for agr AIP genotype II isolates than for isolates representing other agr genotypes (11.4% vs. 18.1%, respectively; P p .02). The presence of dlysin activity was less common in isolates from patients with PB than it was in isolates from patients with RB (71.4% vs. 38.9%, respectively; P p .057) (table 3); this observation suggests that the frequency of agr dysfunction is higher in isolates from patients with PB than it is in isolates from patients with RB. Autolysis. Defective Triton-induced lysis (!30% decrease in OD630 after 5 h of exposure, compared with baseline values) in isolates from patients with PB was not significantly different from that in isolates from patients with RB (23.8% vs. 44.4%, respectively; P p .196), regardless of whether changes in OD630 were compared at 4 h or whether defective Triton-induced lysis was defined as an OD630 change, from the baseline value, of either !40% or !50% (data not shown). Multivariate analyses of patient and bacterial factors. To define potential associations between the outcome of bacteremia and the significant bacterial phenotypic and genotypic characteristics, multivariate logistic regression models were constructed, with PB as the dependent variable and with tPMPsusceptibility profile and PFGE genotype as the independent variables. This effort was largely unsuccessful, because the 2 independent variables were highly correlated with each other. Thus, survival-percentage profiles after exposure to tPMP were significantly higher in the 26 PFGE genotype A isolates than in the 13 PFGE genotype non-A isolates (21.9% 13.5% vs. 8.5% 3.2%, respectively; P p .001). The frequency of PB was also significantly higher in PFGE genotype A isolates than in PFGE genotype non–A isolates (69.2% vs. 23.1%, respectively; P p .015). When the analysis was restricted to patients infected with PFGE genotype A isolates, percentage-survival profiles after exposure to tPMP tended to be higher in the 18 patients (69%) with PB than in the 8 patients (31%) with RB (25.0% 14.4% vs. 14.9% 8.1%, respectively; P p .08). It is noteworthy that 10 (83%) of 12 patients who were infected with isolates having both PFGE genotype A and 120% survival after exposure to tPMP had PB. This rate represents a 7.2-fold– increased risk versus that in the other 27 patients (odds ratio [OR], 7.27 [95% confidence interval {CI}, 1.33–39.9]). These observations suggest that both reduced susceptibility to tPMP in vitro and PFGE genotype A are important factors associated with PB in MRSA isolates. DISCUSSION PB is a poorly understood but increasingly recognized clinical entity. Observational studies have identified clinical and microbiologic risk factors—including occult visceral abscesses, intravascular infections (particularly endocarditis), methicillinresistance, and vancomycin use—that contribute to PB [37]. Nevertheless, there are limited data regarding specific microbial characteristics that influence the interaction between the infecting pathogen, antimicrobial therapy, and innate host defenses that favors the development of PB. In the present study, we analyzed and compared isolates from 2 distinct sets of staphylococcemic patients (i.e., patients with PB and patients with RB) who were selected, on the basis of well-defined clinical and microbiologic criteria, from a large prospective cohort. The 21 patients with PB represented all available cases with documented MRSA bacteremia persisting for 7 days while they were receiving seemingly appropriate antibiotic therapy. For comparison, we randomly selected 18 patients with MRSApositive blood cultures on a single day but with documented bacteremic clearance within the following 4 days. These 2 groups of patients were clinically similar at baseline but experienced different clinical outcomes. In addition to these clinical-outcome differences, we found that these 2 groups were strikingly different in terms of the phenotypic and genotypic characteristics of the isolates from them. MRSA isolates from patients with PB were less likely to produce d-lysin than were isolates from patients with RB. d-Lysin is encoded by the hld locus within the agr operon and is derived from translation of RNA III, the effector molecule of the agr locus [17]. The production of d-lysin can be used as a surrogate marker of agr function [20, 22], and its absence in 170% of PB isolates suggests that agr function is suppressed in these strains. The absence of d-lysin has also been noted in labora Defective Triton lysisa Resolving Persistent bacteremia (n p 21) Note. Data are either no. (%) of isolates in subgroup or mean % SD, unless otherwise indicated. AIP, autoinducing oligopeptide; PFGE, pulsed-field gel electrophoresis; tPMP, thrombin-induced platelet microbicidal protein. a !30% change in OD630 after 5-h exposure to Triton, compared with baseline value. For details and sensitivity analysis using alternative definitions, see the text. tory-derived GISA and hetero-GISA isolates [20–22], as well as in selected clinical-MRSA isolates with the GISA phenotype that have been found in the bloodstream [20]. Other investigators have reported a marked decrease in RNA III transcription in GISA [38, 39]. Compromised function of agr in this setting may be due to significant point mutations [22], dysfunction of coregulatory genes required for agr function, and/ or suppression of agr by upstream regulatory genes (e.g., sigB) [40]. It has been proposed that decreased or complete loss of agr function may serve as one of the multiple steps toward the development of a GISA or hetero-GISA phenotype, especially during exposure to vancomycin [20–22, 41]. Within this context, the presence of MRSA PB during vancomycin therapy may represent an early clinical marker for subsequent evolution of the GISA phenotype. Of particular interest is our recent report showing that, in laboratory-derived and selected clinical MRSA isolates, there is a correlation between exposure to vancomycin, GISA phenotype, agr dysfunction, and low-level resistance to tPMP in vitro [21]. In the present study, PB isolates were significantly less susceptible to tPMP in vitro than were RB isolates. This finding extends our previous observations that reduced susceptibility to tPMP is a common feature in isolates from endovascular infections in general and in IE isolates in particular [5, 10]. Moreover, because PB is more commonly seen in experimental endocarditis caused by tPMP-resistant (vs. tPMP-susceptible) S. aureus strains [11], reduced susceptibility to this innate defense mechanism in clinical isolates may also contribute to PB in staphylococcemic patients. The findings of the present study also underscore the key interactions between exogenous and endogenous antibiotic agents (e.g., vancomycin and tPMP, respectively), in the optimal resolution of clinical endovascular infections (as previously documented in experimental endocarditis [42]). An additional factor that may contribute to MRSA PB involves defective autolysis. Sakoulas et al. have recently confirmed that agr-defective GISA strains derived in vitro are lysed at a slower rate by a membrane detergent than are non-GISA parental strains [20–22]. Other investigators have shown that diminished autolysis is a common feature of clinical GISA strains [43]. Moreover, we have recently shown that the rate of vancomycin-induced lysis in these same GISA strains is also reduced compared with that in non-GISA strains (authors’ unpublished data). It is of interest that normal expression of several key murein-hydrolase genes is agr dependent; thus, agr dysfunction has been noted to cause defective autolysis in S. aureus [44]. In addition, a normally functioning autolytic system appears to contribute to the in vitro staphylocidal pathways of several antimicrobial peptides, including nisin [45, 46] and tPMP [13]. These observations suggest a functional link between agr dysfunction [38, 39], defective autolysis, reduced susceptibility to tPMP, and the GISA phenotype. Dysfunction of agr may also facilitate development of clinical PB in collateral ways. The agr operon normally represses expression of a major S. aureus surface adhesin gene, fnbA, which is overexpressed in the absence of the agr operon [47]. The fnbA adhesin is responsible for S. aureus adhesion to, invasion of, and persistence within endothelial cells, pivotal steps in the pathogenesis of endovascular infection [48–50]. Because vancomycin penetrates poorly within endothelial cells, such intracellular persistence likely enables the organism to evade their antimicrobial effects. Furthermore, production of fnbA leads to endothelial-cell damage and eventual apoptosis [47]. Thus, it is reasonable to postulate that agr dysfunction can foster an intracellular reservoir for the organism, contributing clinically to the development of IE and of PB. In the present study, PFGE analysis indicated that 185% of the isolates from patients with MRSA PB had closely related genotypes likely representing a single genetic lineage. This is particularly noteworthy because the episodes of bacteremia occurred over a period of ∼5 years and included infections with onset occurring in the community, in multiple wards throughout our hospital, and in other hospitals prior to transfer of a patient to our institution. Therefore, this observation is unlikely to represent a specific nosocomial or community-associated spread of a single clone. Previous studies have also observed that particular MRSA chromosomal genotypes are more likely to be associated with PB, prompting speculation that such strains may have an increased tendency to develop the GISA phenotype [41]. In the present study, PB isolates were significantly more likely to exhibit both relative resistance to tPMP in vitro and PFGE genotype A. However, logistic-regression analysis was unable to resolve whether PB was independently associated with one or both of these factors; this result may reflect either that these variables are unrelated or that the sample size or the composition of the data set is relatively limited. The present investigation had potential limitations. There may be selection bias within the study; however, we consider this to be unlikely, because the overall series represents consecutive, prospectively identified patients. Clinical variability was also minimized by the use of rigorous clinical and microbiologic inclusion criteria. Another limitation of the study was that all patients and isolates were obtained from a single institution. Thus, confirmatory investigations using geographically diverse isolates will be necessary to validate our findings. The present study examined distinct groups of patient with MRSA bacteremia who were identified in a large, prospective cohort of patients. One group, designated “PB,” had MRSApositive blood cultures persisting over 7 days despite appropriate antibiotic therapy; the other group, designated “RB,” had rapidly resolving MRSA bacteremia. Although the 2 groups of patients had comparable clinical profiles at baseline, they differed substantially in their clinical courses; in particular, patients with PB had a markedly higher incidence of IE. In vitro studies comparing isolates from the bloodstream of these 2 groups revealed that, compared with RB isolates, PB isolates exhibited (1) significantly lower capacity to be killed in vitro by low levels of tPMP; (2) more-frequent defects in production of d-lysin (suggesting dysfunction within the agr locus); and (3) overrepresentation of a specific PFGE chromosomal genotype. The molecular mechanisms relating these bacterial characteristics to the observed clinical outcomes remain to be defined. References 1. Levine DP , Fromm BS , Reddy BR . Slow response to vancomycin or vancomycin plus rifampin in methicillin-resistant Staphylococcus aureus endocarditis . Ann Intern Med 1991 ; 115 : 674 - 80 . 2. Markowitz N , Quinn EL , Saravolatz LD . Trimethoprim-sulfamethoxazole compared with vancomycin for the treatment of Staphylococcus aureus infection . Ann Intern Med 1992 ; 117 : 390 - 8 . 3. Eng RH , Bishburg E , Smith SM , Scadutto P. Staphylococcus aureus bacteremia during therapy . J Infect Dis 1987 ; 155 : 1331 - 5 . 4. Yeaman MR . The role of platelets in antimicrobial host defense . Clin Infect Dis 1997 ; 25 : 951 - 68 . 5. Bayer AS , Cheng D , Yeaman MR , et al. In vitro resistance to thrombininduced platelet microbicidal protein among clinical bacteremic isolates of Staphylococcus aureus correlates with an endovascular infectious source . Antimicrob Agents Chemother 1998 ; 42 : 3169 - 72 . 6. Bayer AS , Prasad R , Chandra J , et al. In vitro resistance of Staphylococcus aureus to thrombin-induced platelet microbicidal protein is associated with alterations in cytoplasmic membrane fluidity . Infect Immun 2000 ; 68 : 3548 - 53 . 7. Dankert J , Krijgsveld J , van Der WJ , Joldersma W , Zaat SA . Platelet microbicidal activity is an important defense factor against viridans streptococcal endocarditis . J Infect Dis 2001 ; 184 : 597 - 605 . 8. Dhawan VK , Yeaman MR , Cheung AL , Kim E , Sullam PM , Bayer AS . Phenotypic resistance to thrombin-induced platelet microbicidal protein in vitro is correlated with enhanced virulence in experimental endocarditis due to Staphylococcus aureus . Infect Immun 1997 ; 65 : 3293 - 9 . 9. Dhawan VK , Bayer AS , Yeaman MR . In vitro resistance to thrombininduced platelet microbicidal protein is associated with enhanced progression and hematogenous dissemination in experimental Staphylococcus aureus infective endocarditis . Infect Immun 1998 ; 66 : 3476 - 9 . 10. Fowler VG Jr, McIntyre LM , Yeaman MR , et al. In vitro resistance to thrombin-induced platelet microbicidal protein in isolates of Staphylococcus aureus from endocarditis patients correlates with an intravascular device source . J Infect Dis 2000 ; 182 : 1251 - 4 . 11. Kupferwasser LI , Yeaman MR , Shapiro SM , Nast CC , Bayer AS . In vitro susceptibility to thrombin-induced platelet microbicidal protein is associated with reduced disease progression and complication rates in experimental Staphylococcus aureus endocarditis: microbiological, histopathologic, and echocardiographic analyses . Circulation 2002 ; 105 : 746 - 52 . 12. Wu T , Yeaman MR , Bayer AS . In vitro resistance to platelet microbicidal protein correlates with endocarditis source among bacteremic staphylococcal and streptococcal isolates . Antimicrob Agents Chemother 1994 ; 38 : 729 - 32 . 13. Xiong YQ , Yeaman MR , Bayer AS . In vitro antibacterial activities of platelet microbicidal protein and neutrophil defensin against Staphylococcus aureus are influenced by antibiotics differing in mechanism of action . Antimicrob Agents Chemother 1999 ; 43 : 1111 - 7 . 14. Xiong YQ , Bayer AS , Yeaman MR . Inhibition of intracellular macromolecular synthesis in Staphylococcus aureus by thrombin-induced platelet microbicidal proteins . J Infect Dis 2002 ; 185 : 348 - 56 . 15. Yeaman MR , Puentes SM , Norman DC , Bayer AS . Partial purification and staphylocidal activity of thrombin-induced platelet microbicidal protein . Infect Immun 1992 ; 60 : 1202 - 9 . 16. Fujimoto DF , Bayles KW . Opposing roles of the Staphylococcus aureus virulence regulators, Agr and Sar , in Triton X-100- and penicillininduced autolysis. J Bacteriol 1998 ; 180 : 3724 - 6 . 17. Novick RP . Pathogenicity factors and their regulation . In: Fischett VA, Novick RP , Ferretti JJ , Portnoy DA , Rood JI, eds. Washington, DC: American Society for Microbiology, 2000 : 392 - 407 . 18. Jarraud S , Mougel C , Thioulouse J , et al. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease . Infect Immun 2002 ; 70 : 631 - 41 . 19. Jarraud S , Lyon GJ , Figueiredo AM , et al. Exfoliatin-producing strains define a fourth agr specificity group in Staphylococcus aureus . J Bacteriol 2000 ; 182 : 6517 - 22 . 20. Sakoulas G , Eliopoulos GM , Moellering RC Jr, et al. Staphylococcus aureus accessory gene regulator (agr) group II: is there a relationship to the development of intermediate-level glycopeptide resistance ? J Infect Dis 2003 ; 187 : 929 - 38 . 21. Sakoulas G , Eliopoulos GM , Lucindo N , et al. Loss of accessory gene regulator (agr) function and exposure to vancomycin are associated with Staphylococcus aureus resistance to autolysis and thrombin-induced microbicidal proteins in vitro [abstract C1-1741] . In: Programs and Abstracts of the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy (Chicago) . Washington, DC: American Society of Microbiology, 2003 : 94 . 22. Sakoulas G , Eliopoulos GM , Moellering RC Jr, et al. Accessory gene regulator (agr) locus in geographically diverse Staphylococcus aureus isolates with reduced susceptibility to vancomycin . Antimicrob Agents Chemother 2002 ; 46 : 1492 - 502 . 23. Fowler VG Jr, Olsen MK , Corey GR , et al. Clinical identifiers of complicated Staphylococcus aureus bacteremia . Arch Intern Med 2003 ; 163 : 2066 - 72 . 24. Knaus WA , Draper EA , Wagner DP , Zimmerman JE . APACHE II: a severity of disease classification system . Crit Care Med 1985 ; 13 : 818 - 29 . 25. Fowler VG Jr, Kong LK , Corey GR , et al. Recurrent Staphylococcus aureus bacteremia: pulsed-field gel electrophoresis findings in 29 patients . J Infect Dis 1999 ; 179 : 1157 - 61 . 26. National Committee for Clinical Laboratory Standards (NCCLS). Performance standards for antimicrobial susceptibility testing: twelfth informational supplement [M100-S12] . Wayne, PA: NCCLS , 2002 . 27. American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis . Crit Care Med 1992 ; 20 : 864 - 74 . 28. Garner JS , Jarvis WR , Emori TG , Horan TC , Hughes JM . CDC definitions for nosocomial infections , 1988 . Am J Infect Control 1988 ; 16 : 128 - 40 . 29. Libman H , Arbeit RD. Complications associated with Staphylococcus aureus bacteremia . Arch Intern Med 1984 ; 144 : 541 - 5 . 30. Durack DT , Lukes AS , Bright DK. New criteria for diagnosis of infective endocarditis: utilization of specific echocardiographic findings . Duke Endocarditis Service. Am J Med 1994 ; 96 : 200 - 9 . 31. Moore PC , Lindsay JA . Genetic variation among hospital isolates of methicillin-sensitive Staphylococcus aureus: evidence for horizontal transfer of virulence genes . J Clin Microbiol 2001 ; 39 : 2760 - 7 . 32. Ji G , Beavis R , Novick RP . Bacterial interference caused by autoinducing peptide variants . Science 1997 ; 276 : 2027 - 30 . 33. Yeaman MR , Tang YQ , Shen AJ , Bayer AS , Selsted ME . Purification and in vitro activities of rabbit platelet microbicidal proteins . Infect Immun 1997 ; 65 : 1023 - 31 . 34. Tenover FC , Arbeit RD , Goering RV , et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing . J Clin Microbiol 1995 ; 33 : 2233 - 9 . 35. Shimizu A , Berkhoff HA , Kloos WE , George CG , Ballard DN . Genomic DNA fingerprinting, using pulsed-field gel electrophoresis, of Staphylococcus intermedius isolated from dogs . Am J Vet Res 1996 ; 57 : 1458 - 62 . 36. Sneath HA , Sokol RR . Numerical taxonomy: the principles and practice of numerical classification . San Francisco: WH Freeman , 1973 . 37. Khatib R , Johnson LB , Fakih MG , et al. Predictors of persistence (PER) in Staphylococcus aureus bacteremia (SAB): a prospective study [abstract: K-763] . In: Programs and Abstracts of the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy (Chicago) . Washington, DC: American Society of Microbiology, 2003 : 371 . 38. Adhikari RP , Smith JMB , Berger-Bachi B , Cook GM . Decreased oxacillin resistance and RNAIII transcription in Staphylococcus aureus laboratory mutants with decreased susceptibility to vancomycin [abstract C1-1740] . In: Programs and Abstracts of the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy (Chicago) . Washington, DC: American Society of Microbiology, 2003 : 94 . 39. Renzoni A , Francois P , Li D , et al. Modulation of fibronectin-binding proteins (FnBP) expression in a teicoplanin resistant mutant of methicillin-resistant Staphylococcus aureus (MRSA) [abstract C1-1062] . In: Programs and Abstracts of the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy (San Diego). Washington, DC: American Society for Microbiology, 2002 : 71 . 40. Bischoff M and Berger-Bachi B. Teicoplanin stress-selected mutations increasing sigma(B) activity in Staphylococcus aureus . Antimicrob Agents Chemother 2001 ; 45 : 1714 - 20 . 41. Moise-Broder PA , Sakoulas G , Eliopoulos GM , Schentag JJ , Forrest AA , Moellering RC Jr. Association of the accessory gene regulator specificity group II and response of methicillin-resistant Staphylococcus aureus infections to vancomycin [abstract C1-1059] . In: Programs and Abstracts of the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy (San Diego). Washington, DC: American Society for Microbiology, 2002 : 70 . 42. Dhawan VK , Bayer AS , Yeaman MR . Thrombin-induced platelet microbicidal protein susceptibility phenotype influences the outcome of oxacillin prophylaxis and therapy of experimental Staphylococcus aureus endocarditis . Antimicrob Agents Chemother 2000 ; 44 : 3206 - 9 . 43. Boyle-Vavra S , Challapalli M , Daum RS . Resistance to autolysis in vancomycin-selected Staphylococcus aureus isolates precedes vancomycin-intermediate resistance . Antimicrob Agents Chemother 2003 ; 47 : 2036 - 9 . 44. Brunskill EW , Bayles KW . Identification and molecular characterization of a putative regulatory locus that affects autolysis in Staphylococcus aureus . J Bacteriol 1996 ; 178 : 611 - 8 . 45. Bierbaum G , Sahl HG . Induction of autolysis of staphylococci by the basic peptide antibiotics Pep 5 and nisin and their influence on the activity of autolytic enzymes . Arch Microbiol 1985 ; 141 : 249 - 54 . 46. Bierbaum G , Sahl HG . Influence of cationic peptides on the activity of the autolytic endo-beta-N-acetylglucosaminidase of Staphylococcus simulans 22 . FEMS Microbiol Lett 1989 ; 49 : 223 - 7 . 47. Moreillon P , Que YA , Bayer AS . Pathogenesis of streptococcal and staphylococcal endocarditis . Infect Dis Clin North Am 2002 ; 16 : 297 - 318 . 48. Massey RC , Kantzanou MN , Fowler T , et al. Fibronectin-binding protein A of Staphylococcus aureus has multiple, substituting, binding regions that mediate adherence to fibronectin and invasion of endothelial cells . Cell Microbiol 2001 ; 3 : 839 - 51 . 49. Sinha B , Francois PP , Nusse O , et al. Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin alpha5beta1 . Cell Microbiol 1999 ; 1 : 101 - 17 . 50. Sinha B , Herrmann M , Krause KH . Is Staphylococcus aureus an intracellular pathogen? Trends Microbiol 2000 ; 8 : 343 - 4 .


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Vance G. Fowler Jr., George Sakoulas, Lauren M. McIntyre, Venkata G. Meka, Robert D. Arbeit, Christopher H. Cabell, Martin E. Stryjewski, George M. Eliopoulos, L. Barth Reller, G. Ralph Corey, Tiffanny Jones, Natalie Lucindo, Michael R. Yeaman, Arnold S. Bayer. Persistent Bacteremia Due to Methicillin-Resistant Staphylococcus aureus Infection Is Associated with agr Dysfunction and Low-Level In Vitro Resistance to Thrombin-Induced Platelet Microbicidal Protein, Journal of Infectious Diseases, 2004, 1140-1149, DOI: 10.1086/423145