Infective Endocarditis Isolates Research Articles

Differential in vitro susceptibility to ampicillin/ceftriaxone combination therapy among Enterococcus faecalis infective endocarditis clinical isolates.

To investigate the genomic diversity and β-lactam susceptibilities of Enterococcus faecalis collected from patients with infective endocarditis (IE). We collected 60 contemporary E. faecalis isolates from definite or probable IE cases identified between 2018 and 2021 at the University of Pittsburgh Medical Center. We used whole-genome sequencing to study bacterial genomic diversity and employed antibiotic checkerboard assays and a one-compartment pharmacokinetic-pharmacodynamic (PK/PD) model to investigate bacterial susceptibility to ampicillin and ceftriaxone both alone and in combination. Genetically diverse E. faecalis were collected, however, isolates belonging to two STs, ST6 and ST179, were collected from 21/60 (35%) IE patients. All ST6 isolates encoded a previously described mutation upstream of penicillin-binding protein 4 (pbp4) that is associated with pbp4 overexpression. ST6 isolates had higher ceftriaxone MICs and higher fractional inhibitory concentration index values for ampicillin and ceftriaxone (AC) compared to other isolates, suggesting diminished in vitro AC synergy against this lineage. Introduction of the pbp4 upstream mutation found among ST6 isolates caused increased ceftriaxone resistance in a laboratory E. faecalis isolate. PK/PD testing showed that a representative ST6 isolate exhibited attenuated efficacy of AC combination therapy at humanized antibiotic exposures. We find evidence for diminished in vitro AC activity among a subset of E. faecalis IE isolates with increased pbp4 expression. These findings suggest that alternate antibiotic combinations against diverse contemporary E. faecalis IE isolates should be evaluated.

Survey of Bacterial Isolates and Their Antimicrobial Susceptibility Patterns from Dogs with Infective Endocarditis.

Infective endocarditis (IE) is a potentially fatal disease in dogs. Limited information exists regarding the characterization of bacterial isolates from dogs with IE. The objective of this study was to describe bacterial isolates associated with IE and their antimicrobial susceptibility patterns. A retrospective analysis of dogs with IE and bacterial isolates was performed, and antimicrobial susceptibility was interpreted using current veterinary cut points where available. The susceptibility rate was assessed for association with survival and previous antimicrobial administration. Fifty-one bacterial isolates were identified from 45 dogs, and 33 had antimicrobial susceptibility performed. Staphylococcus spp. (14/51; 27.5%) was the most common organism. Antimicrobials with the lowest susceptibility rate were ampicillin (19/26; 73%), doxycycline (16/22; 73%), and enrofloxacin (22/29; 76%) with 12/33 (36%) of isolates exhibiting multidrug resistance (MDR). Individual antimicrobial resistances and the MDR rate were not associated with a difference in survival rate. Bacterial isolates from dogs that had received fluoroquinolone antimicrobials in the month before diagnosis had a higher rate of non-intrinsic fluoroquinolones resistance (5/8;62.5%) compared to those that did not receive fluoroquinolones (2/21; 9.5%) (p = 0.03). Antimicrobial resistance and MDR phenotype were common in this study. Culture and antimicrobial susceptibility testing should be pursued in dogs with IE to help guide antimicrobial therapy.

Dynamics of a Staphylococcus aureus infective endocarditis simulation model.

Infective endocarditis (IE) is a serious infection of the inner surface of heart, resulting from minor lesions in the endocardium. The damage induces a healing reaction, which leads to recruitment of fibrin and immune cells. This sterile healing vegetation can be colonized during temporary bacteremia, inducing IE. We have previously established a novel in vitro IE model using a simulated IE vegetation (IEV) model produced from whole venous blood, on which we achieved stable bacterial colonization after 24 h. The bacteria were organized in biofilm aggregates and displayed increased tolerance toward antibiotics. In this current study, we aimed at further characterizing the time course of biofilm formation and the impact on antibiotic tolerance development. We found that a Staphylococcus aureus reference strain, as well as three clinical IE isolates formed biofilms on the IEV after 6 h. When treatment was initiated immediately after infection, the antibiotic effect was significantly higher than when treatment was started after the biofilm was allowed to mature. We could follow the biofilm development microscopically by visualizing growing bacterial aggregates on the IEV. The findings indicate that mature, antibiotic‐tolerant biofilms can be formed in our model already after 6 h, accelerating the screening for optimal treatment strategies for IE.

ANTIMICROBIAL ACTIVITY OF DALBAVANCIN AGAINST GRAM-POSITIVE BACTERIA ISOLATED FROM PATIENTS WITH INFECTIVE ENDOCARDITIS FROM THE UNITED STATES AND EUROPE (2016-2020): RESULTS FROM THE INTERNATIONAL DALBAVANCIN EVALUATION OF ACTIVITY (IDEA) PROGRAM

TOPIC: Chest Infections TYPE: Original Investigations PURPOSE: The management of infective endocarditis (IE) requires aggressive and prolonged antimicrobial treatment. Dalbavancin (DALBA) has demonstrated potent in vitro activity against gram-positive (GP) organisms commonly responsible for IE and is being evaluated for treatment of complicated bacteremia and IE. METHODS: 16,164 GP organisms were consecutively collected from patients with bloodstream infections (BSIs) in the United States (US; 8,807 isolates from 79 hospitals) and Europe (EU; 7,357 isolates from 42 hospitals in 20 countries) from 2016 to 2020 via the IDEA Program. The collection included 323 isolates from patients with a diagnosis of IE. Isolates were tested for susceptibility (S) against DALBA and comparators by the CLSI broth microdilution method. RESULTS: Among IE isolates, the most common organisms were S. aureus (SA; 52.3%), E. faecalis (EF; 18.0%), and viridans group streptococci (VGS; 10.8%). Among BSI isolates, the most common organisms were SA (45.0%), β-hemolytic streptococci (BHS; 11.0%), and EF (10.8%). DALBA and daptomycin (DAPTO) showed complete activity (100.0%S) against SA from IE, but MIC values from DALBA (MIC50/90, 0.03/0.03 mg/L) were 8- to 16-fold lower than DAPTO (MIC50/90, 0.25/0.5 mg/L). Against SA from IE, linezolid (LZD) and teicoplanin (TEI) were active against 100.0% of isolates and ceftaroline was active against 99.0% of isolates. Vancomycin (VAN) inhibited 98.8% of isolates at ≤1 mg/L. Methicillin-resistant SA (MRSA) rates were 43.3% (US) and 27.5% (EU) among SA from IE (n=169), and 41.1% (US) and 24.0% (EU) among SA from BSI (n=7,279). All EF isolates from IE were S to ampicillin and LZD, whereas 98.3% were S to DALBA (MIC50/90, 0.03/0.06 mg/L), DAPTO (MIC50/90, 0.5/1 mg/L), VAN (MIC50/90, 1/2 mg/L), and TEI (MIC50/90, ≤0.5/≤0.5 mg/L). Against EF, DALBA MIC values were 16- to 32-fold lower than DAPTO and VAN. Among EF from BSI (n=1,748), S to DALBA, DAPTO, and VAN was 98.0%, 99.5%, and 97.7%, respectively. All VGS isolates from IE (n=35) were S to DALBA (at ≤0.12 mg/L), VAN, and LZD; 96.6% of VGS were S to DAPTO. All coagulase-negative staphylococci from IE were S to DALBA (MIC50/90, 0.03/0.12 mg/L; highest MIC, 0.12 mg/L), DAPTO (MIC50/90, 0.25/0.5 mg/L), VAN (MIC50/90, 2/2 mg/L), and LZD (MIC50/90, 1/1 mg/L). E. faecium (EFM) was isolated from 5.6% of IE cases. Among EFM isolates from IE, 66.7% were S to VAN and 72.2% were inhibited at ≤0.12 mg/L of DALBA. BHS was S to DALBA (highest MIC, 0.03 mg/L) and most antimicrobial agents tested. CONCLUSIONS: DALBA exhibited potent in vitro activity against a large collection of contemporary GP isolates recovered from patients with BSI, including IE, in US and EU medical centers. CLINICAL IMPLICATIONS: Due to its prolonged half-life and high potency against GP, DALBA may represent a valuable option for treatment of IE. These results support further investigations on the role of DALBA in the treatment of BSI and IE. DISCLOSURES: my employer received research support relationship with AbbVie Please note: 2020-2022 Added 04/20/2021 by Cecilia Carvalhaes, source=Web Response, value=Grant/Research Support No relevant relationships by Mariana Castanheira, source=Web Response no disclosure on file for Rodrigo Mendes;Research Contractor relationship with Allergan Please note: $1001 - $5000 by Helio Sader, source=Web Response, value=Grant/Research Support Research Contractor relationship with AbbVIe (formerly Allergan) Please note: Ongoing Added 04/15/2021 by Helio Sader, source=Web Response, value=Grant/Research Support No relevant relationships by Streit Streit, source=Web Response

Staphylococcal Enterotoxins and Toxic Shock Syndrome Toxin-1 and Their Association among Bacteremic and Infective Endocarditis Patients in Egypt.

Purpose Infective endocarditis (IE) is a major complication in patients with bacteremia of Staphylococcus (S.) aureus infection. Our aim was to determine the association of the major Staphylococcal superantigens (SAgs), including Staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1), among hospitalized patients diagnosed with bacteremia and those with IE. Methods This study was conducted on 88 patients; of these, 84 (95.5%) had two positive blood cultures. Eighteen out of the 84 patients (21.4%) were diagnosed based on the modified Duke criteria by a cardiologist to have IE. The recovered isolates were screened phenotypically using ELISA followed by molecular analysis of sea, seb, sec, sed, see, and tsst-1, the major SAg coding genes, and the obtained findings were statistically analyzed. Results Phenotypic screening for SE production of 26 selected Staphylococci (15 isolated from the IE patients (10 S. aureus and 5 coagulase negative staphylococci (CoNS)) and 11 from bacteremic patients (10 S. aureus and 1 CoNS)) using ELISA revealed that 12/26 (46%) isolates were SE producers. PCR analysis showed that 19 (73%) isolates were PCR positive for SAg genes with the highest prevalence of the sea gene (79%), followed by seb (63%) and tsst-1 (21%). The least frequent gene was sed (5.3%). Statistical correlations between bacteremic and IE isolates with respect to prevalence of SAgs showed no significant difference (P value = 0.139, effect size = 0.572) indicating no specific association between any of the detected SAgs and IE. Conclusion There is high prevalence of SEs among clinical isolates of Staphylococci recovered from patients suffering bacteremia and those with IE. No significant difference was found among Staphylococcal isolates recovered from patients with bacteremia or IE regarding both phenotypic and genotypic detection of the tested SAgs.

Ceftaroline activity against Staphylococcus aureus isolated from patients with infective endocarditis, worldwide (2010–2019)

ObjectiveTo evaluate the antimicrobial activity of ceftaroline against Staphylococcus aureus isolated from patients with infective endocarditis (IE). Methods23,833 S. aureus isolates were collected consecutively from patients with bloodstream infections (BSI) from 2010 to 2019, via the SENTRY Antimicrobial Surveillance Program, including 396 isolates from patients with a diagnosis of IE. Isolates were collected from 340 medical centers worldwide and susceptibility tested by reference broth microdilution in a monitoring laboratory. ResultsThe oxacillin resistance (MRSA) rate was 29.0% among IE isolates, and was higher in North America (40.3%) than in Europe (25.4%) or the Latin America/Asia-Pacific region (LATAM-APAC; 18.6%). Ceftaroline was active against 95.2% of IE isolates (MIC50/90, 0.25/1 mg/L), with ceftaroline susceptibility higher in North America (99.2%) and LATAM-APAC (98.3%) than in Europe (92.0%). Among MRSA isolates from IE (n = 115; MIC50/90, 1/2 mg/L), ceftaroline susceptibility was 98.0% in North America, 90.9% in LATAM-APAC, and 68.5% in Europe. Among BSI isolates, MRSA rates were 43.6% in North America and 25.6% in Europe, while ceftaroline susceptibility rates were 98.1% in North America and 95.4% in Europe. ConclusionsCeftaroline demonstrated potent in vitro activity against a large collection of S. aureus isolates recovered from patients with BSI, including IE.

ANTIMICROBIAL ACTIVITY OF CEFTAROLINE AGAINST STAPHYLOCOCCUS AUREUS ISOLATED FROM PATIENTS WITH INFECTIVE ENDOCARDITIS WORLDWIDE (2010-2019)

SESSION TITLE: Chest Infections Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: October 18-21, 2020 PURPOSE: Ceftaroline is approved by the United States (US) Food and Drug Administration for community-acquired bacterial pneumonia and skin and skin-structure infections, including cases with concurrent bacteremia, and has demonstrated efficacy in cases of infective endocarditis (IE). We evaluated the antimicrobial activity of ceftaroline against S. aureus isolated from patients with IE. METHODS: 23,834 S. aureus isolates were collected consecutively from patients with bloodstream infections (BSI) from 2010 to 2019 via the SENTRY Antimicrobial Surveillance Program, including 396 isolates patients with a diagnosis of IE. IE isolates were collected from 19 medical centers in the US (n=124), 34 in Europe (EUR; n=213), and 22 in other geographic regions (n=59) and were tested for susceptibility against ceftaroline and comparators by reference broth microdilution method in a monitoring laboratory (JMI Laboratories). RESULTS: Among IE isolates, the overall oxacillin resistance (MRSA) rate was 29.0%, but this rate was higher in the US (40.3%) than EUR (25.4%) and other regions (18.6%). Ceftaroline was active against 95.2% of IE isolates (MIC50/90, 0.25/1 mg/L) and ceftaroline susceptibility was higher in the US (99.2%) and other regions (98.3%) than EUR (92.0%). Among oxacillin-susceptible (MSSA) isolates, the highest ceftaroline MIC value was 0.5 mg/L (94.7% inhibited at ≤0.25 mg/L). Among MRSA isolates from IE (n=115; MIC50/90, 1/2 mg/L), ceftaroline susceptibility was 98.0% in the US and 68.5% in EUR (90.9% in other regions; n=11). The highest ceftaroline MIC was only 2 mg/L and all ceftaroline-nonsusceptible isolates were categorized as susceptible dose dependent or intermediate (0.0% resistant). Dalbavancin (MIC50/90, ≤0.03/0.06 mg/L), daptomycin (MIC50/90, 0.25/0.5 mg/L), linezolid (MIC50/90, 1/2 mg/L), teicoplanin (MIC50/90, ≤2/≤2 mg/L), tigecycline (MIC50/90, 0.06/0.12 mg/L), and vancomycin (MIC50/90, 1/1 mg/L) exhibited complete activity (100.0% susceptible) against S. aureus from IE. Among all BSI S. aureus isolates (n=23,834), MRSA rates were 43.6% in the US and 25.6% in EUR, and ceftaroline susceptibility rates were 98.1% in the US and 95.4% in EUR. CONCLUSIONS: Ceftaroline exhibited potent in vitro activity against a large collection of S. aureus isolates recovered from patients with IE. Ceftaroline was particularly active against MRSA-causing BSI and IE in US medical centers. These results support further investigations to determine the role of ceftaroline in the treatment of IE. CLINICAL IMPLICATIONS: These results support further investigations to determine the role of ceftaroline in the treatment of IE. DISCLOSURES: No relevant relationships by Cecilia Carvalhaes, source=Web Response no disclosure on file for Rodrigo Mendes; Research Contractor relationship with Allergan Please note: $1001 - $5000 Added 03/11/2020 by Helio Sader, source=Web Response, value=Grant/Research Support no disclosure on file for Streit Streit

The microbiological characteristics of Staphylococcus aureus isolated from patients with native valve infective endocarditis

ABSTRACTThe microbiological characteristics of Staphylococcus aureus causing infective endocarditis (IE) have not been investigated thoroughly. We compared the characteristics of S. aureus isolates from patients with and without IE. Cases of S. aureus bacteremia (SAB) were collected from 10 hospitals over 7 years. Cases of native valve IE were matched with non-IE controls according to the following criteria: central-line-associated infection, community-acquired infection, methicillin susceptibility, and if possible, the primary site of infection. Genes coding virulence factors were analyzed using multiplex polymerase chain reactions. Fibrinogen and fibronectin-binding properties were assessed using in vitro binding assays. The fibronectin-binding protein A gene (fnbpA) was sequenced. Of 2,365 cases of SAB, 92 had IE. After matching, 37 pairs of S. aureus isolates from the IE cases and non-IE controls were compared; fnbpA was detected in 91.9% of the IE isolates and 100% of the non-IE isolates (p = 0.24). While the fibrinogen binding ratio was similar (1.07 ± 0.33 vs. 1.08 ± 0.26, p = 0.89), the fibronectin-binding ratio was significantly higher in the IE-group (1.31 ± 0.42 vs. 1.06 ± 0.31, p = 0.01). The proportions of major single-nucleotide polymorphisms in fnbpA were as follows: E652D (2.9% vs. 2.7%), H782Q (65.6% vs. 60.6%), and K786N (65.6% vs. 72.7%). The fibronectin-binding ratio was positively correlated with the number of SNPs present in IE cases (p < 0.001) but not in the non-IE controls (p = 0.124). Fibronectin-binding might play a key role in SAB IE. However, the degree of binding may be mediated by genetic variability between isolates.

Clinical presentation of infective endocarditis caused by different groups of non-beta haemolytic streptococci.

Streptococci are common causes of infective endocarditis (IE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has provided a practical tool for their species determination. We aimed to investigate if particular groups of non-beta heamolytic streptococci were associated with IE or to specific presentations thereof. The Swedish Registry of Infective Endocarditis was used to identify cases of IE caused by streptococci and a local database to identify cases of streptococcal bacteremia. The bacteria were grouped using MALDI-TOF MS and the clinical characteristics of IE caused by different groups were compared. We identified a group of 201 streptococcal IE isolates: 18 isolates belonged to the anginosus, 19 to the bovis, 140 to the mitis, 17 to the mutans, and seven to the salivarius groups. The mitis and mutans groups were significantly more common and the anginosus group less common among IE cases as compared to all cause bacteremia. Patients infected with the bovis group isolates were older, had more cardiac devices, and had more commonly prosthetic valve IE compared to IE caused by streptococci of the other groups. Twenty-one percent of patients needed surgery, and in-hospital mortality was 8% with no significant differences between the groups. Grouping of non-beta haemolytic streptococci using MALDI-TOF MS can provide a basis for decision-making in streptococcal bacteremia. IE caused by bovis group isolates have clinical characteristics distinguishing them from IE caused by other groups of Streptococcus.

Staphylococcus aureus infective endocarditis versus bacteremia strains: Subtle genetic differences at stake

Infective endocarditis (IE)(1) is a severe condition complicating 10–25% of Staphylococcus aureus bacteremia. Although host-related IE risk factors have been identified, the involvement of bacterial features in IE complication is still unclear. We characterized strictly defined IE and bacteremia isolates and searched for discriminant features. S. aureus isolates causing community-acquired, definite native-valve IE (n=72) and bacteremia (n=54) were collected prospectively as part of a French multicenter cohort. Phenotypic traits previously reported or hypothesized to be involved in staphylococcal IE pathogenesis were tested. In parallel, the genotypic profiles of all isolates, obtained by microarray, were analyzed by discriminant analysis of principal components (DAPC)(2). No significant difference was observed between IE and bacteremia strains, regarding either phenotypic or genotypic univariate analyses. However, the multivariate statistical tool DAPC, applied on microarray data, segregated IE and bacteremia isolates: IE isolates were correctly reassigned as such in 80.6% of the cases (C-statistic 0.83, P<0.001). The performance of this model was confirmed with an independent French collection IE and bacteremia isolates (78.8% reassignment, C-statistic 0.65, P<0.01). Finally, a simple linear discriminant function based on a subset of 8 genetic markers retained valuable performance both in study collection (86.1%, P<0.001) and in the independent validation collection (81.8%, P<0.01). We here show that community-acquired IE and bacteremia S. aureus isolates are genetically distinct based on subtle combinations of genetic markers. This finding provides the proof of concept that bacterial characteristics may contribute to the occurrence of IE in patients with S. aureus bacteremia.

Clinical MRSA isolates from skin and soft tissue infections show increased in vitro production of phenol soluble modulins

Phenol-soluble modulins (PSMs) are amphipathic, pro-inflammatory proteins secreted by most Staphylococcus aureus isolates. This study tested the hypothesis that invitro PSM production levels are associated with specific clinical phenotypes. 177 methicillin-resistant S. aureus (MRSA) isolates from infective endocarditis (IE), skin and soft tissue infection (SSTI), and hospital-acquired/ventilator-associated pneumonia (HAP) were matched by geographic origin, then genotyped using spa-typing. Invitro PSM production was measured by high performance liquid chromatography/mass spectrometry. Statistical analysis was performed using Chi-squared or Kruskal-Wallis tests as appropriate. Spa type 1 was significantly more common in SSTI isolates (62.7% SSTI; 1.7% IE; 16.9% HAP; p<0.0001) while HAP and IE isolates were more commonly spa type 2 (0% SSTI; 37.3% IE; 40.7% HAP; p<0.0001). USA300 isolates produced the highest levels of PSMs invitro. SSTI isolates produced significantly higher quantities of PSMα1-4, PSMβ1, and δ-toxin than other isolates (p<0.001). These findings persisted when USA300 isolates were excluded from analysis. Increased invitro production of PSMs is associated with an SSTI clinical source. This significant association persisted after exclusion of USA300 genotype isolates from analysis, suggesting that PSMs play a particularly important role in the pathogenesis of SSTI as compared to other infection types.

Rise of CC398 Lineage of Staphylococcus aureus among Infective Endocarditis Isolates Revealed by Two Consecutive Population-Based Studies in France

Staphylococcus aureus isolates from two prospective studies on infective endocarditis (IE) conducted in 1999 and 2008 and isolated from non-IE bacteremia collected in 2006 were spa-typed and their virulence factors were analyzed with a microarray. Both populations were genetically diverse, with no virulence factors or genotypes significantly more associated with the IE isolates compared with the non-IE isolates. The population structure of the IE isolates did not change much between 1999 and 2008, with the exception of the appearance of CC398 methicillin-susceptible Staphylococcus aureus (MSSA) isolates responsible for 5.6% of all cases in 2008. In 1999, this lineage was responsible for no cases. The increasing prevalence of S. aureus in IE is apparently not the result of a major change in staphylococcal population structure over time, with the exception of the emerging CC398 MSSA lineage.

Porcine and Human Community Reservoirs ofEnterococcusfaecalis, Denmark

To the Editor: Enterococcus faecalis, which exists commensally in the gut in warm-blooded animals and humans, is an opportunistic pathogen that causes a variety of community-acquired and health care–associated infections, such as urinary tract and intraabdominal infections, bacteremia, and endocarditis (1). Only a few studies have assessed the relationships between clinical E. faecalis strains; strains endemic to the health care setting; and community strains residing in humans, animals, or animal-origin food (2). Recently we showed that the emergence of high-level gentamicin-resistant (HLGR) E. faecalis among patients with infective endocarditis (IE) coincided with an increase in HLGR E. faecalis in the pig population in Denmark (3). The majority of isolates belonged to the same clonal group (sequence type [ST] 16), suggesting that pigs constitute a community reservoir of HLGR E. faecalis. We investigated human and porcine community reservoirs of other E. faecalis clonal types associated with IE in humans in Denmark. A total of 20 consecutive gentamicin-susceptible E. faecalis isolates were obtained from IE patients in North Denmark Region during 1996–2002 (Table A1). Cases of IE were classified as definite (n = 12) or possible (n = 8) according to the modified Duke criteria (4). A case of community-acquired E. faecalis infection (n = 6) was defined in accordance with strict criteria applied for methicillin-resistant Staphylococcusaureus (5); otherwise, cases were deemed to be health care associated (n = 14) (Table A1). HLGR ST16 isolates recovered from 2 IE patients during the study period have been characterized (3) and were excluded from the present study. Using multilocus sequence typing (6), we identified 14 STs among the 20 IE isolates (Table A1), then compared them with STs from 2 collections of E. faecalis isolates collected as part of the Danish Integrated Antimicrobial Resistance Monitoring and Research Program (www.danmap.org): 1) all 14 isolates recovered from community-dwelling humans in North Denmark Region during 2002–2006 with approval from the local ethics committee ([KF] 01-006/02), which were classified into 10 STs in this study (Table A1); and 2) 19 pig isolates from 2001 that were shown in a previous study to belong to 12 STs (7). Among the 14 STs identified in IE isolates, 4 (ST19, ST21, ST72, and ST306) and 2 (ST40 and ST97) were also found among isolates from community-dwelling humans and pigs, respectively (Table A1). Isolates belonging to these 6 STs were further characterized by pulsed-field gel electrophoresis (PFGE) by using SmaI and grouped into PFGE pulsotypes as described (3). STs and PFGE pulsotypes (A–F) were largely concordant (ST97:A, ST72:B, ST19:C, ST40:D, ST21:E, and ST306:F), except for 2 isolates belonging to ST72 and ST40, for which PFGE banding patterns (U1 and U2, respectively) were unrelated to the major PFGE pulsotypes (A–F), and 1 ST306 isolate exhibiting the ST21-like PFGE banding pattern E (Table A1). These findings confirm the genetic relatedness of IE isolates with those from community-dwelling humans (ST72:B, ST19:C, ST21:E, and ST306:F) and pigs (ST97:A and ST40:D). Seven (64%) of 11 IE isolates belonging to these 6 clonal types originated from IE patients with health care–associated risk factors (Table A1), which suggests that health care users are predisposed to colonization and infection with E. faecalis strains residing in human and porcine community reservoirs. Previous reports have shown that epidemiologically distinct E. faecalis populations differ in terms of biofilm formation, virulence gene content, and antimicrobial drug susceptibility profiles (2,8). Therefore, we characterized all isolates with respect to these traits. Isolates were categorized into strong, medium, weak, and nonbiofilm formers by using the method of Mohamed et al. (8). The presence of 12 virulence-associated and pathogenicity island genes (ebpA, gelE, ef1824, hylA, ef1896, ef2347, ef2505, hylB, ace, cbh, esp, and ef0571) was investigated by using colony lysates and probes that have been described elsewhere (9). The antimicrobial drug susceptibility profiles (ampicillin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, kanamycin, linezolid, penicillin, streptomycin, teicoplanin, tetracycline, and vancomycin) were determined by the Sensititre system (Trek Diagnostic Systems, East Grinstead, UK) in accordance with Clinical and Laboratory Standards Institute guidelines (10). The isolates were generally homogenous within each clonal type in terms of biofilm formation, presence of virulence-associated and pathogenicity island genes, and resistance profiles (Table A1), further supporting that IE isolates are genetically related to those from community-dwelling humans and pigs, respectively. Notably, most IE isolates were susceptible to ampicillin (100%), penicillin (100%), vancomycin (100%), high-level gentamicin (100%), and high-level streptomycin (80%), which are the drugs of choice in therapeutic regiments for E. faecalis endocarditis. In conclusion, our results suggest that the normal intestinal microflora of humans and pigs are community reservoirs of clinical E. faecalis and link 2 porcine-origin clonal types of gentamicin-susceptible E. faecalis, ST97:A, and ST40:D to IE in humans in Denmark. This finding strengthens existing evidence that pigs can be a source of serious infections in humans.

Methicillin-Susceptible Staphylococcus aureus Endocarditis Isolates Are Associated With Clonal Complex 30 Genotype and a Distinct Repertoire of Enterotoxins and Adhesins

Using multinational collections of methicillin-susceptible Staphylococcus aureus (MSSA) isolates from infective endocarditis (IE) and soft tissue infections (STIs), we sought to (1) validate the finding that S. aureus in clonal complex (CC) 30 is associated with hematogenous complications and (2) test the hypothesis that specific genetic characteristics in S. aureus are associated with infection severity. IE and STI isolates from 2 cohorts were frequency matched by geographic origin. Isolates underwent spa typing to infer CC and multiplex polymerase chain reaction for presence of virulence genes. 114 isolate pairs were genotyped. IE isolates were more likely to be CC30 (19.5% vs 6.2%; P = .005) and to contain 3 adhesins (clfB, cna, map/eap; P < .0001 for all) and 5 enterotoxins (tst, sea, sed, see, and sei; P ≤ .005 for all). CC30 isolates were more likely to contain cna, tst, sea, see, seg, and chp (P < .05 for all). MSSA IE isolates were significantly more likely to be CC30 and to possess a distinct repertoire of virulence genes than MSSA STI isolates from the same region. The genetic basis of this association requires further study.

The role of streptococcal plasmin(ogen) binding in infective endocarditis

The ability of viridans group streptococci (VGS) to bind human plasminogen and its subsequent activation into plasmin may contribute to the pathogenesis of streptococcal endocarditis. The increased proteolytic activity acquired through cell-bound plasmin may lead to a decreased stability of the streptococcal vegetation and possible embolisation. Twenty-two infective endocarditis isolates and 16 non-infective endocarditis isolates were screened for their ability to bind plasminogen through the quantification of its active form plasmin, using the colorimetric substrate D-Val-Leu-Lys p-nitroanilide. The species of the VGS assessed expressed a universal capability to bind human plasminogen, although they did so with differing affinities and independently of the site of isolation.

Heterogeneous Vancomycin‐Intermediate Susceptibility Phenotype in Bloodstream Methicillin‐ResistantStaphylococcus aureusIsolates from an International Cohort of Patients with Infective Endocarditis: Prevalence, Genotype, and Clinical Significance

The significance of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) is unknown. Using a multinational collection of isolates from methicillin-resistant S. aureus (MRSA) infective endocarditis (IE), we characterized patients with IE with and without hVISA, and we genotyped the infecting strains. MRSA bloodstream isolates from 65 patients with definite IE from 8 countries underwent polymerase chain reaction (PCR) for 31 virulence genes, pulsed-field gel electrophoresis, and multilocus sequence typing. hVISA was defined using population analysis profiling. Nineteen (29.2%) of 65 MRSA IE isolates exhibited the hVISA phenotype by population analysis profiling. Isolates from Oceania and Europe were more likely to exhibit the hVISA phenotype than isolates from the United States (77.8% and 35.0% vs 13.9%; P < .001). The prevalence of hVISA was higher among isolates with a vancomycin minimum inhibitory concentration of 2 mg/L (P = .026). hVISA-infected patients were more likely to have persistent bacteremia (68.4% vs 37.0%; P = .029) and heart failure (47.4% vs 19.6%; P = .033). Mortality did not differ between hVISA- and non-hVISA-infected patients (42.1% vs 34.8%, P = .586). hVISA and non-hVISA isolates were genotypically similar. In these analyses, the hVISA phenotype occurred in more than one-quarter of MRSA IE isolates, was associated with certain IE complications, and varied in frequency by geographic region.

Fibronectin‐binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgG binding to the FcγRIIa receptor

Staphylococcus aureus is a leading cause of infective endocarditis (IE). Platelet activation promoted by S. aureus resulting in aggregation and thrombus formation is an important step in the pathogenesis of IE. Here, we report that the fibrinogen/fibronectin-binding proteins FnBPA and FnBPB are major platelet-activating factors on the surface of S. aureus from the exponential phase of growth. Truncated derivatives of FnBPA, presenting either the fibrinogen-binding A domain or the fibronectin-binding BCD region, each promoted platelet activation when expressed on the surface of S. aureus or Lactococcus lactis, indicating two distinct mechanisms of activation. FnBPA-promoted platelet activation is mediated by fibrinogen and fibronectin bridges between the A domain and the BCD domains, respectively, to the low affinity form of the integrin GPIIb/IIIa on resting platelets. Antibodies recognizing the FnBPA A domain or the complex between the FnBPA BCD domains and fibronectin were essential for activation promoted by bacteria expressing the A domain or the BCD domain respectively. Activation was inhibited by a monoclonal antibody (IV-3) specific for the FcgammaRIIa IgG receptor on platelets. We propose that the activation of quiescent platelets by bacteria expressing FnBPs involves the formation of a bridge between the bacterial cell and the platelet surface by (i) fibronectin and fibrinogen interacting with the low affinity form of GPIIb/IIIa and (ii) by antibodies specific to FnBPs that engage the platelet Fc receptor FcgammaRIIa. Platelet activation by S. aureus clinical IE isolates from both the exponential and stationary phases of growth was completely inhibited by monoclonal antibody IV-3 suggesting that the IgG-FcgammaRIIa interaction is of fundamental importance for platelet activation mediated by this organism. This suggests new avenues for development of therapeutics against vascular infections.

Pathogenic potential of lactobacilli

Lactobacilli are often considered to be commensal or beneficial participants in human microbial ecology and considerable research is being carried out into the effects of the use of lactobacilli as additives in both human and animal diets. However, lactobacilli also cause some human diseases (e.g. dental caries, rheumatic vascular disease, septicaemia and infective endocarditis (IE)), and have recently been identified as potential emerging pathogens in elderly and immunocompromised patients, particularly those receiving broad spectrum antibiotic therapy. The identification of potential pathogenic traits amongst lactobacilli will therefore facilitate the use of the organisms for probiotic purposes. The ability to aggregate human platelets is considered to be a possible pathogenic trait in the progression of IE. A comparison of bacterial cell surface properties amongst L. rhamnosus strains showed that platelets were aggregated by 5 5 IE strains and 8 16 laboratory strains. For the L. paracasei subsp. paracasei strains the respective numbers were 2 5 and 2 9 . However two strains, morphological mutants of a non-aggregating strain, which had been re-isolated after passaging through rats were found to aggregate platelets. No loss of aggregating function occured on extensive subculturing of IE strains. Aggregation also occurred with 11 14 strains for five other species, namely, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus oris, Lactobacillus plantarum and Lactobacillus salivarius, with each species being represented indicating that the property is not uncommon in the genus. A comparison of IE and oral isolates of L. rhamnosus and L. paracasei subsp. paracasei and seven other Lactobacillus species, has shown that the binding of both fibronectin and fibrinogen by lactobacilli is greatly increased, up to 50 fold, when the pH is reduced from 7.0 to 5.0. Re-exposing the lactobacilli to a neutral pH environment releases most of the bound proteins, but the amount still remaining bound to the cell is several times more than is bound at neutral pH. Lactobacilli will also bind to the proteins that make up the extracellular matrix of endothelial cells. Lactobacilli bound significantly better to collagen types I and V than to types III and IV ( p<0.01). Further, strains isolated from IE cases, particularly L. rhamnosus strains, bound significantly better to types I and V than did ‘normal’ strains ( p<0.02). Type V collagen has been demonstrated at the sites of endothelial damage. Thus the binding of lactobacilli, particularly L. rhamnosus to these collagen types may be of importance in the early stages of colonization of the damaged heart valve. The research therefore suggests that lactobacilli being considered for probiotic purposes should be screened for these potentially pathogenic traits.

In vitro resistance to platelet microbicidal protein correlates with endocarditis source among bacteremic staphylococcal and streptococcal isolates

Traditionally, platelets have been thought to contribute to the induction and propagation of infective endocarditis (IE). However, recent studies suggest that platelets may potentially mitigate IE via secretion of alpha-granule-derived platelet microbicidal protein (PMP). In this study, we compared the PMP susceptibility of bacteremic isolates from patients with and without IE. Isolates of Staphylococcus aureus (n = 17), coagulase-negative staphylococci (CNS; n = 28), viridans streptococci (VS; n = 54), and Enterococcus faecalis (n = 20), each at a final inoculum of 2 x 10(3) CFU/ml, were exposed to PMP [100 U/ml, (5 micrograms/ml)] for 2 h, and the percent survival was determined. For S. aureus, CNS, and VS isolates, there was a significant correlation between an IE source and increased percent survival post-PMP exposure; the mean percent survivals of S. aureus, CNS, and VS were significantly greater for IE versus non-IE isolates (P < 0.005 for each organism). No significant correlation was observed between the source of bacteremic E. faecalis isolates and PMP susceptibility. These data suggest that staphylococcal and VS (but not enterococcal) resistance to PMP may facilitate either the induction or progression of IE.