Agr Dysfunction Research Articles

Profiling daptomycin resistance among diverse methicillin-resistant Staphylococcus aureus lineages in China.

Daptomycin (DAP) is effective against methicillin-resistant Staphylococcus aureus (MRSA). However, reduced susceptibility to DAP in MRSA may lead to treatment failures. We aim to determine the distribution of DAP minimum inhibitory concentrations (MICs) and DAP heteroresistance (hDAP) among MRSA lineages in China. A total of 472 clinical MRSA isolates collected from 2015 to 2017 in China were examined for DAP susceptibility. All isolates (n = 472) were found to be DAP susceptible, but 35.17% (166/472) of them exhibited a high DAP MIC (MIC >0.5 µg/mL). The high DAP MIC group contained a larger proportion of isolates with a higher vancomycin or teicoplanin MIC (>1.5 µg/mL) than the low DAP MIC group (19.3% vs 7.8%, P < 0.001; 22.3% vs 8.2%, P < 0.001). We compared the clonal complex (CC) distributions and clinical characteristics in MRSA isolates stratified by DAP MIC. CC5 isolates were less susceptible to DAP (MIC50 = 1 µg/mL) than CC59 isolates (MIC50 = 0.5 µg/mL, P < 0.001). Population analysis profiling revealed that 5 of 10 ST5 and ST59 DAP-susceptible MRSA isolates investigated exhibited hDAP. The results also showed that CC5 MRSA with an agrA mutation (I238K) had a higher DAP MIC than those with a wild-type agrA (P < 0.001). The agrA-I238K mutation was found to be associated with agr dysfunction as indicated by the loss of δ-hemolysin production. In addition, agr/psmα defectiveness was associated with hDAP in MRSA. Whole-genome sequencing analysis revealed mutations in mprF and walR/walK in DAP-resistant subpopulations, and most DAP-resistant subpopulations (6/8, 75%) were stable. Our study suggests that the increased DAP resistance and hDAP in MRSA may threaten the effectiveness against MRSA infections.

Genotypes of Staphylococcus aureus Clinical Isolates Are Associated with Phenol-Soluble Modulin (PSM) Production

Phenol-soluble modulins (PSMs) are important S. aureus virulence factors that cause cytolysis, mast cell degranulation, and stimulate inflammatory responses. In this study, PSM production by S. aureus clinical isolates was measured by liquid chromatography/mass spectrometry (LC-MS) and correlated with staphylococcal protein A (spa) type and staphylococcal cassette chromosome mec (SCCmec) type. Of 106 S. aureus clinical isolates, 50 (47.2%) corresponded to methicillin-susceptible S. aureus (MSSA) and 56 (52.8%) to methicillin-resistant S. aureus (MRSA). LC-MS analysis revealed no significant difference in average PSMα3, PSMα4, PSMβ2, and δ-toxin production between MSSA and MRSA isolates, but PSMα1, PSMα2, and PSMβ1 production were higher in MSSA than MRSA. This study demonstrated that average PSMα1–α4, PSMβ1–β2, and δ-toxin production by SCCmec type II strains was significantly lower than the IV, IVA, and V strains. Most of the SCCmec type II strains (n = 17/25; 68.0%) did not produce δ-toxin, suggesting a dysfunctional Agr system. The spa type t111 (except one strain) and t2460 (except one strain producing PSM α1–α4) did not produce PSMα1–α4 and δ-toxin, while average PSM production was higher among the t126 and t1784 strains. This study showed that the genotype of S. aureus, specifically the spa and SCCmec types, is important in characterizing the production of PSMs.

Lung-Adapted Staphylococcus aureus Isolates With Dysfunctional Agr System Trigger a Proinflammatory Response.

Staphylococcus aureus dominates the lung microbiota of children with cystic fibrosis (CF) and persistent clones are able to establish chronic infection for years, having a direct deleterious impact on lung function. However, in this context, the exact contribution of S. aureus to the decline in respiratory function in children with CF is not elucidated. To investigate the contribution of persistent S. aureus clones in CF disease, we undertook the analysis of sequential isogenic isolates recovered from 15 young CF patients. Using an air-liquid infection model, we observed a strong correlation between S. aureus adaption in the lung (late isolates), low toxicity, and proinflammatory cytokine secretion. Conversely, early isolates appeared to be highly cytotoxic but did not promote cytokine secretion. We found that cytokine secretion was dependent on staphylococcal protein A (Spa), which was selectively expressed in late compared to early isolates as a consequence of dysfunctional agr quorum-sensing system. Finally, we demonstrated the involvement of TNF-α receptor 1 signaling in the inflammatory response of airway epithelial cells to these lung-adapted S. aureus isolates. Our results suggest an unexpected direct role of bacterial lung adaptation in the progression of chronic lung disease by promoting a proinflammatory response through acquired agr dysfunction.

Direct Quantitative Immunochemical Analysis of Autoinducer Peptide IV for Diagnosing and Stratifying Staphylococcus aureus Infections.

An immunochemical strategy to detect and quantify AIP-IV, the quorum sensing (QS) signaling molecule produced by Staphylococcus aureusagr type IV, is reported here for the first time. Theoretical calculations and molecular modeling studies have assisted on the design and synthesis of a suitable peptide hapten (AIPIVS), allowing to obtain high avidity and specific antibodies toward this peptide despite its low molecular weight. The ELISA developed achieves an IC50 value of 2.80 ± 0.17 and an LOD of 0.19 ± 0.06 nM in complex media such as 1/2 Tryptic Soy Broth. Recognition of other S. aureus AIPs (I–III) is negligible (cross-reactivity below 0.001%), regardless of the structural similarities. A pilot study with a set of clinical isolates from patients with airways infection or colonization demonstrates the potential of this ELISA to perform biomedical investigations related to the role of QS in pathogenesis and the association between dysfunctional agr or the agr type with unfavorable clinical outcomes. The AIP-IV levels could be quantified in the low nanomolar range in less than 1 h after inoculating agr IV-genotyped isolates in the culture broth, while those genotyped as I–III did not show any immunoreactivity after a 48 h growth, pointing to the possibility to use this technology for phenotyping S. aureus. The research strategy here reported can be extended to the rest of the AIP types of S. aureus, allowing the development of powerful multiplexed chips or point-of-care (PoC) diagnostic devices to unequivocally identify its presence and its agr type on samples from infected patients.

Prevalence and genetic characteristic of Staphylococcus aureus with agr dysfunction

Prevalence and genetic characteristic of Staphylococcus aureus with agr dysfunction

C-reactive protein predicts persistent bacteremia caused by community-acquired methicillin-resistant Staphylococcus aureus strain.

There is limited data on persistent bacteremia (PB) caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). Here, we aimed to investigate the clinical and microbiological characteristics of PB caused by the major CA-MRSA strain in Korea (ST72-SCCmecIV). All adult patients with S. aureus bacteremia were prospectively investigated from August 2008 to December 2018. Patients with ST72 MRSA bacteremia were included in the study. Patients were stratified into the PB group (defined as positive blood cultures for ≥ 3days) and short bacteremia (SB) group. A total of 291 patients were included, comprising 115 (39.5%) with PB and 176 (60.5%) with SB. Although the 30-day mortality did not differ between PB and SB, recurrent bacteremia within 12weeks was significantly more common in PB (8.7% vs 1.7%; P = 0.01). Multivariate analysis showed risk factors of PB were liver cirrhosis (adjusted odds ratio [aOR], 3.27; 95% confidence interval [CI], 1.50-7.12), infective endocarditis (aOR, 7.13; 95% CI, 1.37-37.12), bone and joint infections (aOR, 3.76; 95% CI, 1.62-8.77), C-reactive protein ≥ 10mg/dL (aOR, 2.20; 95% CI, 1.22-3.95), metastatic infection (aOR, 7.35; 95% CI, 3.53-15.29), and agr dysfunction (aOR, 2.47; 95% CI, 1.05-5.81). PB occurred in approximately 40% of bacteremia caused by ST72 MRSA with a significantly higher recurrence rate. Patients with risk factors of PB, including liver cirrhosis, high initial CRP, infective endocarditis, or bone and joint infections, might require early aggressive treatment.

Varied Contribution of Phospholipid Shedding From Membrane to Daptomycin Tolerance in Staphylococcus aureus.

It has been suggested that daptomycin can be inactivated by lipids released by Staphylococcus aureus and that this effect is antagonized by phenol soluble modulins (PSMs), which bind to the shed lipids. PSM production is regulated by the Agr system, and others have shown that loss of the Agr function enhances S. aureus survival in the presence of daptomycin. Here we assessed the impact of Agr function on daptomycin activity and lipid metabolism under various conditions. Daptomycin activity was evaluated against three sets of isogenic strain series with wild-type or dysfunctional Agr using static daptomycin time-kills over 24 h and against one strain pair using in vitro pharmacokinetic/pharmacodynamic (PK/PD) models simulating clinical daptomycin exposure for 48 h. We performed comprehensive lipidomics on bacterial membranes and the spent media to correlate lipid shedding with survival. In static time-kill experiments, two agr-deficient strains (SH1000- and USA300 LAC ΔagrA) showed improved survival for 8 h compared with their corresponding wild-type strains as seen in previous studies, but this difference did not persist for 24 h. However, four other agr-deficient strains (SH1001 and JE2 agr KOs) did not demonstrate improved survival compared to isogenic wild-type strains at any time in the time-kills. Lipidomics analysis of SH1000, SH1001, and SH1000- strains showed daptomycin exposure increased lipid shedding compared to growth controls in all strains with phosphatidylglycerols (PGs), lysylPGs and cardiolipins predominating. In the cell pellets, PGs and lysylPGs decreased but cardiolipins were unchanged with daptomycin exposure. The shed lipid profiles in SH1001 and SH1000- were similar, suggesting that the inability to resist daptomycin by SH1001 was not because of differences in lipid shedding. In the PK/PD model, the agr mutant SH1000- strain did not show improved survival relative to SH1000 either. In conclusion, inactivation of daptomycin by shed lipids may be dependent on genetic background, the specific agr mutations, or the techniques used to generate these KOs rather than the overall function of the Agr system, and its contribution to daptomycin tolerance seems to be varied, transient, and growth-condition dependent.

1186. Cefazolin inoculum effect predicts reduced susceptibility to other antibiotics and patient outcomes in MSSA endovascular infections

BackgroundMSSA Infective endocarditis (IE) is inherently a high-burden infection with up to a 30% mortality rate. Cefazolin is an appealing treatment option for IE with low toxicity and a favorable dosing scheme. However, cefazolin has been associated with treatment failure in IE, attributed to an inoculum effect. The specific mechanism underlying the cefazolin inoculum effect (CIE) remains undetermined, but CIE has been linked to both blaZ expression and agr dysfunction. This study aims to determine whether CIE is linked to reduced susceptibility to other antibiotics and worse outcomes regardless of therapy in MSSA endovascular infections.MethodsSixty-four MSSA strains were collected from patients with endovascular infections not treated with cefazolin. To determine CIE phenotype, strains were cultured and MICs assayed for cefazolin, nafcillin, and vancomycin at 107 CFU/mL for high-inocula (HI) and 105 CFU/mL for standard-inocula (SI). This study defined CIE as a ≥ 4-fold increase in MIC at HI compared to SI, with at least an MIC of 4 mg/L at HI. Nitrocefin disks identified blaZ expression, and beta lysin disks were used to determine hemolysin type and agr function. Patient outcomes of mortality and bacteremia duration were assessed across cohorts.ResultsTwenty-four strains exhibit a CIE (38%), with 10 strains having an MIC of ≥ 32mg/L at HI. Nafcillin and vancomycin also had an inoculum effect, uncoupled from the CIE and occurring at a lower frequency and amplitude at HI. Presence of CIE had a greater association with blaZ expression (71% vs 25%) than agr dysfunction (38% vs 20%). 50% (9/18) of CIE infections were cleared within 48 hours while 77% (20/26) of CIE-negative infections were cleared within 48 hours (P=0.106). However, presence of CIE was not associated with increased mortality (25% CIE-positive vs 35%; P=0.578)ConclusionPrevious studies for CIE failed to enrich for isolates from endovascular sources, where inocula are known to be high. This study presents one of the largest endovascular source cohorts for CIE evaluation. It identifies that CIE prevalence (38%) is higher than reports from diverse infection sources (10-36%). CIE appears to predict bacteremia duration with other MSSA treatment options, suggesting mechanisms independent of blaZ and agr function for this phenomenon.Disclosures Warren Rose, PharmD, MPH, Merck (Grant/Research Support)Paratek (Grant/Research Support)

269. Clinical Characteristics and Outcomes of Persistent Staphylococcus aureus Bacteremia

Background Staphylococcus aureus bacteremia (SAB) is a leading cause of bacteremia and persistent SAB is associated with poor outcomes. We evaluated key clinical characteristics and outcomes associated with persistent SAB.MethodsWe reviewed patients enrolled in a prospective cohort of adult patients with S. aureus bacteremia at a tertiary hospital from August 2008 to December 2018. Clinical characteristics, outcomes, and microbiologic characteristics of patients with persistent bacteremia (≥ 3 d) were evaluated.ResultsOf the total 969 patients, 617 (63.7%) patients had persistent bacteremia. The median duration of bacteremia with persistent bacteremia was 5 days. The most common sources of persistent bacteremia were central venous catheter-related infection (33.4%) and bone and joint infection (14.9%). Methicillin resistant S. aureus (MRSA) isolates were analyzed in 372 (60.3%) patients and metastatic infections were 217 (35.2%) with persistent bacteremia. In the multivariate analysis, APACHE Ⅱ score (adjusted odds ratio [aOR], 1.07; 95% confidence interval [CI], 1.03–1.10), Charlson comorbidity index score (aOR, 1.14; 95% CI, 1.04–1.25), liver cirrhosis (aOR, 2.47; 95% CI, 1.44–4.23), and S. aureus pneumonia (aOR, 3.04; 95% CI, 1.29–7.18) were independently associated with 30-d mortality. In persistent MRSA bacteremia, ST5-SCCmecⅡ was 59.7% (222/372) and agr dysfunction was 64.8% (241/372). After adjusting for confounding factors, APACHE Ⅱ score (aOR, 1.08; 95% confidence interval [CI], 1.04–1.12), liver cirrhosis (aOR, 3.09; 95% CI, 1.56–6.14), and S. aureus pneumonia (aOR, 4.37; 95% CI, 1.40–13.67) were independently associated with 30-d mortality.Table 1. Demographic and Clinical characteristics of Patients With Persistent Bacteremia Table 2. Microbiologic Characteristics and Genotypes in MRSA Isolates Responsible for Persistent Bacteremia Fig 1. Duration of Staphylococcus aureus bacteremia ConclusionIn persistent bacteremia, clinical factors, including APACHE Ⅱ score, Charlson comorbidity index score, liver cirrhosis, and S. aureus pneumonia contribute to 30-d mortality.Disclosures All Authors: No reported disclosures

Dysfunctional accessory gene regulator (agr) as a prognostic factor in invasive Staphylococcus aureus infection: a systematic review and meta-analysis

The accessory gene regulator (agr) locus of Staphylococcus aureus is a quorum-sensing virulence regulator. Although there are many studies concerning the effect of dysfunctional agr on the outcomes of S. aureus infection, there is no systematic review to date. We systematically searched for clinical studies reporting outcomes of invasive S. aureus infections and the proportion of dysfunctional agr among their causative strains, and we performed a meta-analysis to obtain estimates of the odds of outcomes of invasive S. aureus infection with dysfunctional versus functional agr. Of 289 articles identified by our research strategy, 20 studies were meta-analysed for crude analysis of the impact of dysfunctional agr on outcomes of invasive S. aureus infection. Dysfunctional agr was generally associated with unfavourable outcomes (OR 1.32, 95% CI 1.05–1.66), and the impact of dysfunctional agr on outcome was more prominent in invasive methicillin-resistant S. aureus (MRSA) infections (OR 1.54, CI 1.20–1.97). Nine studies were meta-analysed for the impact of dysfunctional agr on the 30-day mortality of invasive S. aureus infection. Invasive MRSA infection with dysfunctional agr exhibited higher 30-day mortality (OR 1.40, CI 1.03–1.90) than that with functional agr. On the other hand, invasive MSSA infection with dysfunctional agr exhibited lower 30-day mortality (OR 0.51, CI 0.27–0.95). In the post hoc subgroup analysis by the site of MRSA infection, dysfunctional agr was associated with higher 30-day mortality in MRSA pneumonia (OR 2.48, CI 1.17–5.25). The effect of dysfunctional agr on the outcome of invasive S. aureus infection may vary depending on various conditions, such as oxacillin susceptibility and the site of infection. Dysfunctional agr was generally associated with unfavourable clinical outcomes and its effect was prominent in MRSA and pneumonia. Dysfunctional agr may be applicable for outcome prediction in cases of invasive MRSA infection with hardly eradicable foci such as pneumonia.

Vancomycin MIC and agr dysfunction in invasive MRSA infections in southern Brazil.

In methicillin-resistant Staphylococcus aureus (MRSA) treatment, the vancomycin minimum inhibitory concentration (MIC) increase, vancomycin heteroresistance (hVISA) presence, and accessory gene regulator (agr) dysfunction are predictors of vancomycin therapy failure. This study evaluated the association between vancomycin MIC (≥ 1.0μg/mL) and agr dysfunction in invasive MRSA isolates. Vancomycin MIC, hVISA phenotype, agr group, and function were determined in 171 MRSA isolates obtained between 2014 and 2019 from hospitals in Porto Alegre, Brazil. All MRSA were susceptible to vancomycin; 16.4% of these had MIC ≥ 1.0μg/mL. Seventeen MRSA isolates expressed the hVISA phenotype; 35.3% of them had MIC of 1.5μg/mL. agr groups I (40.9%) and II (47.1%) were the most found groups for MRSA and hVISA isolates, respectively. The proportion of MRSA with vancomycin MIC ≥ 1.0μg/mL in agr group II was significantly higher than in agr groups I and III (p = 0.002). agr dysfunction was observed in 4.7% (8/171) of MRSA, especially those with vancomycin MIC ≥ 1.0μg/mL (p < 0.001). In addition, six isolates (35.3%; 6/17) with hVISA phenotype presented agr dysfunction, which was significantly higher than that in non-hVISA phenotype (p < 0.001). In conclusion, agr dysfunction in MRSA is associated with vancomycin MIC ≥ 1.0μg/mL and hVISA phenotype, which suggests that agr dysfunction might confer potential advantages on MRSA to survive in invasive infections.

Effects of subinhibitory concentrations of chlorhexidine and mupirocin on biofilm formation in clinical meticillin-resistant Staphylococcus aureus

The effects of subinhibitory concentrations (sub-MICs) of antibacterial agents on the biofilm-forming ability of Staphylococcus aureus require further study. To investigate the effects of sub-MICs of chlorhexidine and mupirocin on biofilm formation in clinical meticillin-resistant Staphylococcus aureus (MRSA) isolates. MRSA isolates were collected from patients with bloodstream infections at a tertiary care hospital. The basal level of biofilm formation and biofilm induction by sub-MICs of chlorhexidine and mupirocin were evaluated by measuring biofilm mass stained with Crystal Violet. Of the 112 MRSA isolates tested, 63 (56.3%) and 44 (39.3%) belonged to sequence type (ST)5 and ST72 lineages, respectively, which are the predominant healthcare- and community-associated clones in South Korea. ST5 isolates were more likely to have chlorhexidine MIC ≥4 (73.0% vs 29.5%), resistance to mupirocin (23.8% vs 0%), agr dysfunction (73.0% vs 9.1%), and qacA/B gene (58.7% vs 2.3%) compared to ST72 isolates. The basal level of biofilm formation ability was frequently stronger in ST72 isolates compared to ST5 isolates (77.3% vs 12.7%). Sub-MICs of chlorhexidine and mupirocin promoted biofilm formation in 56.3% and 53.6%, respectively, of all isolates. Biofilm induction was more prevalent in ST5 isolates (85.7% for chlorhexidine, 69.8% for mupirocin) than in ST72 isolates (15.9% for chlorhexidine, 27.3% for mupirocin). Sub-MICs of chlorhexidine and mupirocin promoted biofilm formation in half of the clinical MRSA isolates. Our results suggest that ST5 MRSA biofilm can be induced together with some other bacterial virulent factors following exposure to chlorhexidine, which might confer a survival advantage to this clone in the healthcare environment.

Intracellular Environment and agr System Affect Colony Size Heterogeneity of Staphylococcus aureus.

Staphylococcus aureus causes chronic and relapsing infections, which may be difficult to treat. So-called small colony variants (SCVs) have been associated with chronic infections and their occurrence has been shown to increase under antibiotic pressure, low pH and intracellular localization. In clinics, S. aureus isolated from invasive infections often show a dysfunction in the accessory gene regulator (agr), a major virulence regulatory system in S. aureus. To assess whether intracellular environment and agr function influence SCV formation, an infection model was established using lung epithelial cells and skin fibroblasts. This allowed analyzing intracellular survival and localization of a panel of S. aureus wild type strains and their isogenic agr knock out mutants as well as a natural dysfunctional agr strain by confocal laser scanning microscopy (CLSM). Furthermore, bacterial colonies were quantified after 1, 3, and 5 days of intracellular survival by time-lapse analysis to determine kinetics of colony appearance and SCV formation. Here, we show that S. aureus strains with an agr knock out predominantly resided in a neutral environment, whereas wild type strains and an agr complemented strain resided in an acidic environment. S. aureus agr mutants derived from an intracellular environment showed a higher percentage of SCVs as compared to their corresponding wild type strains. Neutralizing acidic phagolysosomes with chloroquine resulted in a significant reduction of SCVs in S. aureus wild type strain 6850, but not in its agr mutant indicating a pH dependent formation of SCVs in the wild type strain. The in-depth understanding of the interplay between intracellular persistence, agr function and pH should help to identify new therapeutic options facilitating the treatment of chronic S. aureus infections in the future.

Association between biomass formation and the prognosis of infective endocarditis due to Staphylococcus aureus

IntroductionThe aim of this study was to evaluate the association between biomass formation and the clinical characteristics and prognosis of Staphylococcus aureus infective endocarditis (IE). MethodsWe prospectively studied 209 S. aureus strains causing IE. Biomass formation was examined using the crystal violet assay and quantified spectrophotometrically. The average (SD) optical density of the biomass was compared for each clinical, microbiological (methicillin-resistance, vancomycin MIC≥1.5μg/ml) and molecular (clonal complex, agr type and agr dysfunction) variable according to their presence or absence. The primary clinical endpoints studied were in-hospital death, severe sepsis, persistent bacteraemia, symptomatic peripheral embolisms and prosthetic valve IE. ResultsMean age was 66.1 years, 61.5% of patients were male and the median age-adjusted Charlson comorbidity index was 5 points (IQR 3–8). In-hospital mortality was 37.3%. Strains belonging to CC5 and CC22 had optical biomass densities [mean (SD) 1.573 (1.14) vs 0.942 (0.98) p<0.001 and 1.720 (0.94) vs 1.028 (1.04) p=0.001, respectively]. Strains belonging to CC5 and CC22 had significantly higher optical biomass densities [1.369 (1.18) vs 0.920 (0.93) p=0.008]. No statistically significant differences were found in the clinical endpoints studied. ConclusionsHigh biomass production was associated with CC5 and CC22 but not with higher hospital mortality, septic complications, type of endocarditis, methicillin-resistance, elevated vancomycin MIC or agr dysfunction.

Long-term methicillin-resistant Staphylococcus aureus bacteremia persisting for more than 2 weeks: risk factors and outcomes.

The clinical significance of long-term methicillin-resistant Staphylococcus aureus (MRSA) bacteremia remains unclear. We evaluated the clinical, microbiological characteristics, and clinical outcomes of long-term MRSA bacteremia. A nested case-control study was conducted in a prospective cohort of adult patients with MRSA bacteremia at a tertiary hospital between August 2008 and December 2017. Patients with long-term MRSA bacteremia (≥ 14days) were compared with control patients, defined as having bacteremia that resolved in less than 3days. The following variables were documented: heteroresistance phenotype, genotypes, agr dysfunction, and the presence of 41 virulence genes in isolates. Of the total 890 patients studied, 69 patients (7.8%) exhibited long-term MRSA bacteremia and 599 (67.3%) exhibited resolving bacteremia. The most common sources of long-term bacteremia were central venous catheter-related infection (39%) and osteomyelitis (19%). Independent risk factors for long-term MRSA bacteremia included male sex (adjusted odds ratio [aOR] = 2.43), community-acquired bacteremia (aOR = 2.93), the presence of a prosthetic device (aOR = 3.40), and osteomyelitis (aOR = 7.98). Metastatic infections developed more frequently in patients with long-term bacteremia than in those with resolving bacteremia (56.5% vs. 8.0%; P < 0.001). Although there were no significant differences in 30-day, 12-week, or in-hospital mortality rates between the two groups, infection-attributable mortality was higher in the long-term bacteremia group (23.2% vs. 11.5%; P = 0.01). Microbiological characteristics did not differ significantly between the two groups. Clinical factors, including community-acquired bacteremia, the presence of a prosthetic device, and osteomyelitis, appear to contribute to long-term MRSA bacteremia more than microbiological factors.

Association between biomass formation and the prognosis of infective endocarditis due to Staphylococcus aureus

IntroductionThe aim of this study was to evaluate the association between biomass formation and the clinical characteristics and prognosis of Staphylococcus aureus infective endocarditis (IE). MethodsWe prospectively studied 209 S. aureus strains causing IE. Biomass formation was examined using the crystal violet assay and quantified spectrophotometrically. The average (SD) optical density of the biomass was compared for each clinical, microbiological (methicillin-resistance, vancomycin MIC≥1.5μg/ml) and molecular (clonal complex, agr type and agr dysfunction) variable according to their presence or absence. The primary clinical endpoints studied were in-hospital death, severe sepsis, persistent bacteraemia, symptomatic peripheral embolisms and prosthetic valve IE. ResultsMean age was 66.1 years, 61.5% of patients were male and the median age-adjusted Charlson comorbidity index was 5 points (IQR 3–8). In-hospital mortality was 37.3%. Strains belonging to CC5 and CC22 had optical biomass densities [mean (SD) 1.573 (1.14) vs 0.942 (0.98) p<0.001 and 1.720 (0.94) vs 1.028 (1.04) p=0.001, respectively]. Strains belonging to CC5 and CC22 had significantly higher optical biomass densities [1.369 (1.18) vs 0.920 (0.93) p=0.008]. No statistically significant differences were found in the clinical endpoints studied. ConclusionsHigh biomass production was associated with CC5 and CC22 but not with higher hospital mortality, septic complications, type of endocarditis, methicillin-resistance, elevated vancomycin MIC or agr dysfunction.

596. Distinct, Segregated Daptomycin-Susceptible and Daptomycin-Nonsusceptible Staphylococcus aureus Populations Associated with Tricuspid-Valve Infective Endocarditis

BackgroundLoss of daptomycin susceptibility in Staphylococcus aureus is often associated with sequestered foci of infection, driven by selection pressure from both administered antibiotics and host defense peptides. Susceptibility testing of the organism cultured from blood is assumed to parallel that of the infectious foci, such as heart valves. We studied a case of tricuspid valve endocarditis where one leaflet yielded exclusively daptomycin-nonsusceptible S. aureus and another leaflet yielded purely daptomycin-susceptible S. aureus. We examined the responses of the two populations to different anti-staphylococcal therapies to identify regimens effective against both isolates.MethodsBoth isolates were whole-genome-sequenced using Illumina technologies. The presence of heterogeneous daptomycin-resistant subpopulations was assessed by dilution plating and population analysis profiling. One compartment pharmacokinetic/pharmacodynamic modeling was used to simulate different potential antistaphylococcal pharmacotherapies against each isolate. Hemolysin activity was evaluated as a surrogate for accessory gene regulator function.ResultsThe daptomycin-susceptible isolate did not demonstrate heteroresistance while the daptomycin-resistant population was uniformly daptomycin nonsusceptible. The daptomycin non-susceptible isolate demonstrated regrowth by 72 hours of simulated treatment with vancomycin (2 g Q12H) or daptomycin (10 mg/kg daily). Adding cefazolin (2 g Q8H) to vancomycin or daptomycin prevented regrowth at 72 hours. The daptomycin-resistant isolate was deficient in hemolysin production suggesting agr dysfunction. Comparative sequencing identified daptomycin-resistant isolate mutations in mprF, purR and agrA.ConclusionThis case underscores the complex dynamics of the emergence of S. aureus resistance to daptomycin in vivo. Our pharmacokinetic modeling supports combination therapy in the treatment of endovascular MRSA infection. Reduced hemolytic activity supports the hypothesis that agr modulation is associated with persistent infection and/or treatment failure. Ongoing studies will identify features of distinct bacterial populations that promote ecological succession during infection at a sequestered anatomical site.Disclosures All authors: No reported disclosures.

Accessory gene regulator (agr) dysfunction was unusual in Staphylococcus aureus isolated from Chinese children

BackgroundStaphylococcus aureus (S. aureus) with accessory gene regulator (agr) dysfunction occurs in health care settings. This study evaluated the prevalence and the molecular and drug resistance characteristics of S. aureus with dysfunctional agr in a pediatric population in Beijing, China.ResultsA total of 269 nonduplicate S. aureus clinical isolates were isolated from Beijing Children’s Hospital, including 211 methicillin-resistant S. aureus (MRSA) from September 2010–2017 and 58 methicillin-sensitive S. aureus (MSSA) from February 2016–2017. Only 8 MRSA and 2 MSSA isolates were identified as agr dysfunction, and the overall prevalence rate was 3.7%. For MRSA isolates, ST59-SCCmec IV and ST239-SCCmec III were the most common clones, and the prevalence rate of agr dysfunction in ST239-SCCmec III isolates (17.39%) was significantly higher than in ST59-SCCmec IV (1.69%) and other genotype strains (P = 0.006). Among the agr dysfunctional isolates, only one MRSA ST59 isolate and one MSSA ST22 isolate harbored pvl. No significant difference was detected between agr dysfunction and agr functional isolates regarding the biofilm formation ability (P = 0.4972); however, 9/10 agr dysfunctional isolates could effectuate strong biofilm formation and multidrug resistance. Among MRSA, the non-susceptibility rates to ciprofloxacin, gentamicin, and trimethoprim-sulfamethoxazole were significantly higher in agr dysfunctional isolates than in isolates with functional agr (P < 0.05). Two isolates belonging to ST239 had no mutations in agr locus, but a synonymous mutation was found in agrA in another ST239 isolate. The inactivating mutations were detected in other seven agr dysfunctional isolates. The variants were characterized by non-synonymous changes (n = 5) and frameshift mutations (insertions, n = 2), which mainly occurred in agrC and agrA.ConclusionsThe results showed that agr dysfunctional S. aureus was not common in Chinese children, and ST59-SCCmec IV was associated with lower prevalence of agr dysfunction as compared to ST239-SCCmec III isolates. The agr dysfunctional isolates were healthcare-associated, multidrug resistant and form strong biofilm, which suggested that agr dysfunction might offer potential advantages for S. aureus to survive in a medical environment.

The Cefazolin Inoculum Effect and the Presence of type A blaZ Gene according to agr Genotype in Methicillin-Susceptible Staphylococcus aureus Bacteremia.

BackgroundRecent data suggests the inoculum effect of methicillin-susceptible Staphylococcus aureus (MSSA) against beta-lactam antibiotics and their association with functionality or genotypic variation of agr locus.MethodsMSSA blood isolates were collected at a tertiary care hospital in Korea from June 2014 to December 2017. The functionality of the agr operon was measured by δ-hemolysin assays. Multiplex PCR was performed to determine the agr genotype. The cefazolin minimum inhibitory concentrations (MICs) at a high inoculum concentration (~5 × 107 CFU/ml) were compared to the MICs at a standard inoculum concentration (~5 × 105 CFU/ml) to identify strains with the cefazolin inoculum effect (CIE). The DNA sequencing of blaZ gene was performed to classify the blaZ genotype.ResultsAmong the 195 MSSA blood isolates, agr genotype I was most common (68.2%), followed by type III (16.4%), type IV (9.2%), and type II (6.2%). Sixty-seven (34.3%) MSSA isolates had dysfunctional agr, but neither CIE nor blaZ genotype was associated with dysfunctional agr. The MSSA with agr type III genotype exhibited significantly higher CIE positivity (agr III 43.8% vs. non-agr III 5.5%, P <0.01) and erythromycin/clindamycin resistance. In the subgroup analysis of type A blaZ possessing MSSA, almost all of the agr III MSSA isolates exhibited CIE, while only 20% of non-agr III isolates had CIE (P <0.01).ConclusionIn MSSA blood isolates, CIE might be associated with agr genotype rather than with dysfunctional agr.

The Microevolution and Epidemiology of Staphylococcus aureus Colonization during Atopic Eczema Disease Flare

Staphylococcus aureus is an opportunistic pathogen and variable component of the human microbiota. A characteristic of atopic eczema (AE) is colonization by S. aureus, with exacerbations associated with an increased bacterial burden of the organism. Despite this, the origins and genetic diversity of S. aureus colonizing individual patients during AE disease flares is poorly understood. To examine the microevolution of S. aureus colonization, we deep sequenced S. aureus populations from nine children with moderate to severe AE and 18 non-atopic children asymptomatically carrying S. aureus nasally. Colonization by clonal S. aureus populations was observed in both AE patients and control participants, with all but one of the individuals carrying colonies belonging to a single sequence type. Phylogenetic analysis showed that disease flares were associated with the clonal expansion of the S. aureus population, occurring over a period of weeks to months. There was a significant difference in the genetic backgrounds of S. aureus colonizing AE cases versus controls (Fisher exact test, P = 0.03). Examination of intra-host genetic heterogeneity of the colonizing S. aureus populations identified evidence of within-host selection in the AE patients, with AE variants being potentially selectively advantageous for intracellular persistence and treatment resistance.