Study of Staphylococcus aureus pathogenic genes by transfer and expression in the less virulent organism Streptococcus gordonii

Because Staphylococcus aureus strains contain multiple virulence factors, studying their pathogenic role by single-gene inactivation generated equivocal results. To circumvent this problem, we have expressed specific S. aureus genes in the less virulent organism Streptococcus gordonii and tested the recombinants for a gain of function both in vitro and in vivo. Clumping factor A (ClfA) and coagulase were investigated. Both gene products were expressed functionally and with similar kinetics during growth by streptococci and staphylococci. ClfA-positive S. gordonii was more adherent to platelet-fibrin clots mimicking cardiac vegetations in vitro and more infective in rats with experimental endocarditis (P < 0.05). Moreover, deleting clfA from clfA-positive streptococcal transformants restored both the low in vitro adherence and the low in vivo infectivity of the parent. Coagulase-positive transformants, on the other hand, were neither more adherent nor more infective than the parent. Furthermore, coagulase did not increase the pathogenicity of clfA-positive streptococci when both clfA and coa genes were simultaneously expressed in an artificial minioperon in streptococci. These results definitively attribute a role for ClfA, but not coagulase, in S. aureus endovascular infections. This gain-of-function strategy might help solve the role of individual factors in the complex the S. aureus-host relationship.

[Antibiotic prophylaxis for late blood-borne infections of joint prostheses]

By analogy with endocarditis prophylaxis, patients with joint prostheses are often given antibiotics before invasive procedures or dental treatment. However, this analogy is not justified: The pathogenesis and bacterial spectrum of infections of artificial joints differ from those of endocarditis. Since the efficacy of administering prophylactic antibiotics to patients with joint prostheses has never been scientifically proven, there is no general indication for such prophylaxis. On the other hand, infections in other parts of the body should be actively sought and treated promptly. Prophylactic antibiotic administration may be appropriate in individual cases during a procedure in patients who are at increased risk of a haematogenic prosthesis infection as a result of bacteraemia. For operations routinely performed under perioperative antibiotic cover, the same prophylaxis should also be used for patients with joint prostheses.

Influence of the developmental state of valvular lesions on the antimicrobial activity of cefotaxime in experimental enterococcal infections

Cefotaxime has little antimicrobial activity in vitro against most strains of enterococci, as measured by conventional MICs and MBCs. However, the MICs of cefotaxime against many enterococci are markedly reduced by the addition of serum to the test medium. To assess the relevance of this observation in vivo, we examined the efficacy of cefotaxime in experimental Streptococcus faecalis endocarditis. Since response to antimicrobial agents may vary with the degree of vegetation development, therapeutic efficacy was assessed both in rabbits with newly formed vegetations and in rabbits with well-developed endocardial lesions. Peak serum levels of cefotaxime (50.1 +/- 20.0 micrograms/ml) exceeded the MIC in medium supplemented with serum (4 micrograms/ml), but not in Mueller-Hinton broth alone (greater than 64 micrograms/ml). After 4 days of therapy, animals with newly formed lesions (therapy initiated 1 h after infection, transvalvular catheters removed) had lower mean vegetation bacterial titers than did untreated controls. Among animals with mature vegetations (therapy initiated 12 h after infection, catheters indwelling), the rate of mortality was significantly reduced by cefotaxime therapy. However, no difference in vegetation titers was observed. Thus, cefotaxime demonstrated antienterococcal activity within newly formed vegetations, but did not inhibit bacterial proliferation within well-established vegetations.

[Periodontitis and cardiovascular disease]

The prevalence of periodontitis and cardiovascular disease (CVD) is high. A mixed infectious biofilm etiology of periodontitis is known but not fully established in CVD. Cofactors; smoking habits, stress, ethnicity, genetics, socioeconomics and age contribute to both diseases. The objectives of this report are to summarize factors in regards to CVD and periodontitis that are clinically relevant. The hypothesis behind a relationship between the two conditions can be founded in (I) shared infections etiology, (II) shared inflammatory response, (III) epidemiological and case-control studies, and (IV) periodontal studies demonstrating improvements of CVD markers. Streptococcus species in the S. mitis group, and S. anginosus group have been identified in periodontitis and are known as pathogens in endocarditis possibly transported from the oral cavity to the heart through bacteremia during dental therapies, and tooth brushing. Other periodontal bacteria such as Porphyromonas gingivalis, Fusobacterium nucleatum and Parvimonas micra are beta-lactamase producing and may contribute to antibiotic resistance (extended spectrum beta-lactamases). Other bacteria in CVD and periodontitis include Staphylococcus aureus, and Pseudomonas aeruginosa. Chlamydia pneumoniae and P. gingivalis lipopolyysaccharide capsels share homology and induce heat-shock protein activity and a cascade of proinflammatory cytokines. Associations between periodontitis and CVD have been presented in many studies when controlling for confounders. Other studies have demonstrated that periodontal therapies increase brachial artery flow rate and reduce serum inflammatory cytokine levels. Thus, physicians caring for subjects at CVD risk should consult with dentists/periodontists. Dentists must improve their medical knowledge and also learn to consult with physicians when treating patients at CVD risk.

Significance of Staphylococcus lugdunensis bacteremia: report of 28 cases and review of the literature

BACKGROUND: Staphylococcus lugdunensis endocarditis has been associated with an aggressive course. The aim of this study was to determine factors associated with the development of endocarditis in patients with S. lugdunensis bacteremia. METHODS: A retrospective analysis of all patients with S. lugdunensis bacteremia in three tertiary care centers in Switzerland was performed. Data regarding medical history, symptoms, and susceptibility of S. lugdunensis isolates were collected. Our results were reviewed in the context of the current literature. RESULTS: A total of 28 patients with S. lugdunensis bacteremia were identified. Of the 13 patients with endocarditis, all were community acquired. Cardiac surgery was performed in 85% of these patients; mortality was 23%, reflecting the aggressive course of this disease. In contrast, in the 15 patients without endocarditis, no complications associated with S. lugdunensis bacteremia were observed. In 73%, a probable source was identified in the form of a venous catheter or other foreign device. Only three of these episodes were community acquired. No difference was observed in susceptibility of the S. lugdunensis isolates to penicillin, which was 77% in endocarditis isolates, and 87% in isolates of bacteremia without endocarditis, respectively. CONCLUSION: S. lugdunensis bacteremia is associated with endocarditis in up to 50% of patients. Every patient with community-acquired S. lugdunensis bacteremia should be carefully examined for signs of endocarditis. Once S. lugdunensis endocarditis is diagnosed, close monitoring is essential and surgical treatment should be considered early. In the nosocomial setting, endocarditis is far less frequent, and S. lugdunensis bacteremia is usually associated with a catheter or other foreign materials.

Deleterious outcome of No-React-treated stentless valved conduits after aortic root replacement: why were warnings ignored?

OBJECTIVE: The implantation of a composite graft is the treatment of choice for patients with aortic root disease if the valve cannot be preserved and the patient is not a suitable candidate for a Ross procedure. Several years ago, the Shelhigh NR-2000C (Shelhigh, Inc, Millburn, NJ) was introduced in Europe. Being a totally biologic conduit and considering the lack of homografts, the graft seemed an ideal conduit for patients with destructive endocarditis, as well as for older patients who were not suitable candidates for oral anticoagulation. METHODS: From 2001 until 2006, the Shelhigh NR-2000C stentless valved conduit was implanted in 115 patients for various aortic root pathologies. The conduit consists of a bovine pericardial straight graft with an incorporated porcine stentless valve. Aortic root repair was performed during standard cardiopulmonary bypass and mild hypothermia in the majority of patients. Deep hypothermic circulatory arrest combined with selective antegrade cerebral perfusion was used when the repair extended into the arch. RESULTS: Seven patients with uncomplicated early outcome presented with unexpected sudden disastrous findings at the level of the aortic root, although 1-year follow-up computed tomographic scans were normal. Four of these patients underwent emergency operations because of desintegration of the graft, along with rupture of the aortic root. Retrospectively, the main findings were persistent fever or subfebrility over months and a halo-like enhancement on computed tomographic scans. Extensive microbiologic examinations were performed without finding a causative organism. CONCLUSION: The use of the Shelhigh aortic stentless conduit can no longer be advocated, and meticulous follow-up of patients in whom this device has been implanted has to be recommended.

The Ross procedure in infants and young children

BACKGROUND: This study reviews our experience with the Ross procedure in infants and young children. METHODS: From September 1993 to September 2004, 52 children less than 15 years of age underwent a Ross procedure. The patients ranged in age from 4 days to 15 years old (median, 5 years). Fifteen patients (29%) were less than 2 years of age. The predominant indication for the Ross procedure was aortic stenosis. Sixteen patients underwent a Ross-Konno procedure for severe left ventricular outflow tract obstruction. Thirty-four patients had 48 previous interventions. Preoperatively, 6 patients showed severe left ventricular dysfunction, and 2 of the patients required ventilation and inotropic support. Concomitant procedures were performed in 8 patients. Three patients had a mitral valve replacement, 2 patients had a ventricular septal defect closure and an aortic arch reconstruction, 2 patients had aortic arch reconstructions, and 1 patient had resection of a coarctation and a ventricular septal defect closure. RESULTS: Patients were followed up for a median of 43 months (range, 1 to 130). Overall survival was 85% +/- 5% at 1 and 82% +/- 5% at 2, 5, and 10 years. Hospital mortality was 5 of 52 patients (9.6%). All deaths occurred in neonates or infants less than 2 months of age, who needed urgent surgery. Three patients died late of noncardiac causes. At last follow-up, all patients were classified in New York Heart Association functional class I or II. No patient had endocarditis of the autograft or the right ventricular outflow tract replacement. During the follow-up, no event of thrombembolism was observed. No patient required the insertion of a permanent pacemaker. Overall freedom from reoperation is 57% +/- 15% at 10 years. One patient required the replacement of the autograft at 6 months postoperatively. The development of mild aortic insufficiency was observed in 24 patients, and moderate aortic insufficiency in 1 patient during follow-up. Freedom from reoperation for the right ventricular outflow tract replacement is 60% +/- 15% at 10 years. CONCLUSIONS: The Ross procedure represents an attractive approach to aortic valve disease in young children. However, a high early mortality rate has to be considered when performing this procedure in neonates or infants who present in critical preoperative condition.

[Does yoghurt gnaw at cardiac valves?]

A 22-year-old man with pre-existing aortic valve disease contracted acute lactobacillus endocarditis six weeks after a dental procedure despite adequate prophylaxis. We discuss the limitations of prophylaxis for infective endocarditis in use until the end of 2008 and describe the new updated guidelines. We also explain the treatment of lactobacillus endocarditis and speculate on possible health risks of the increasing use of lactobacillus-containing dairy products, especially in immune-compromised patients.

[Fever of unknown origin as a sign of calcium pyrophosphate deposition disease]

Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease may manifest clinically as septic fever (40 degrees C), acute pseudogout attack of knee, wrist and shoulders, or as a variety of patterns of chronic inflammatory or degenerative joint disease. The association of pseudogout with fever is less widely recognised and may lead to over-investigation, delay in appropriate treatment and disproportionate costs. We report on a 67-year-old woman with a history of recurrent episodes of fever and polyarthritis every 2 months for the last 3 years. Because of this she was hospitalised several times, finally with suspected culture-negative endocarditis, and was treated for 6 weeks with gentamicin, rifampicin and vancomycin. During this therapy the patient again developed septic fever and acute arthritis of the right wrist. Radiographs of the wrist, knee and symphysis pubis revealed prominent chondrocalcinosis and destructive arthropathy.

Adherence to host extracellular matrix and serum components by Enterococcus faecium isolates of diverse origin.

Enterococcus faecium has emerged as an important cause of nosocomial infections over the last two decades. We recently demonstrated collagen type I (CI) as a common adherence target for some E. faecium isolates and a significant correlation was found to exist between acm-mediated CI adherence and clinical origin. Here, we evaluated 60 diverse E. faecium isolates for their adherence to up to 15 immobilized host extracellular matrix and serum components. Adherence phenotypes were most commonly observed to fibronectin (Fn) (20% of the 60 isolates), fibrinogen (17%) and laminin (Ln) (13%), while only one or two of the isolates adhered to collagen type V (CV), transferrin or lactoferrin and none to the other host components tested. Adherence to Fn and Ln was almost exclusively restricted to clinical isolates, especially the endocarditis-enriched nosocomial genogroup clonal complex 17 (CC17). Thus, the ability to adhere to Fn and Ln, in addition to CI, may have contributed to the emergence and adaptation of E. faecium, in particular CC17, as a nosocomial pathogen.

Characterization of the ebp(fm) pilus-encoding operon of Enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection.

We recently identified 15 genes encoding putative surface proteins with features of MSCRAMMs and/or pili in the Enterococcus faecium TX0016 (DO) genome, including four predicted pilus-encoding gene clusters; we also demonstrated that one of these, ebpABC(fm), is transcribed as an operon, that its putative major pilus subunit, EbpC(fm) (also called pilB), is polymerized into high molecular weight complexes, and that it is enriched among clinical E. faecium isolates. Here, we created a deletion of the ebpABC(fm) operon in an endocarditis-derived E. faecium strain (TX82) and showed, by a combination of whole-cell ELISA, flow cytometry, immunoblot and immunogold electron microscopy, that this deletion abolished EbpC(fm) expression and eliminated EbpC(fm)-containing pili from the cell surface. However, transcription of the downstream sortase, bps(fm), was not affected. Importantly, the ebpABC(fm) deletion resulted in significantly reduced biofilm formation (p < 0.0001) and initial adherence (p < 0.0001) versus the wild-type; both were restored by complementing ebpABC(fm) in trans, which also restored cell surface expression of EbpC(fm) and pilus production. Furthermore, the deletion mutant was significantly attenuated in two independent mixed infection mouse urinary tract experiments, i.e., outnumbered by the wild-type in kidneys (p = 0.0003 and < 0.0001, respectively) and urinary bladders (p = 0.0003 and = 0.002). In conclusion, we have shown that the ebpABC(fm) locus encodes pili on the E. faecium TX82 cell surface and provide the first evidence that pili of this emerging pathogen are important for its ability to form biofilm and to cause infection in an ascending UTI model.

The hylEfm gene in pHylEfm of Enterococcus faecium is not required in pathogenesis of murine peritonitis.

BACKGROUND: Plasmids containing hylEfm (pHylEfm) were previously shown to increase gastrointestinal colonization and lethality of Enterococcus faecium in experimental peritonitis. The hylEfm gene, predicting a glycosyl hydrolase, has been considered as a virulence determinant of hospital-associated E. faecium, although its direct contribution to virulence has not been investigated. Here, we constructed mutants of the hylEfm-region and we evaluated their effect on virulence using a murine peritonitis model. RESULTS: Five mutants of the hylEfm-region of pHylEfmTX16 from the sequenced endocarditis strain (TX16 [DO]) were obtained using an adaptation of the PheS* system and were evaluated in a commensal strain TX1330RF to which pHylEfmTX16 was transferred by mating; these include i) deletion of hylEfm only; ii) deletion of the gene downstream of hylEfm (down) of unknown function; iii) deletion of hylEfm plus down; iv) deletion of hylEfm-down and two adjacent genes; and v) a 7,534 bp deletion including these four genes plus partial deletion of two others, with replacement by cat. The 7,534 bp deletion did not affect virulence of TX16 in peritonitis but, when pHylEfmTX16Δ7,534 was transferred to the TX1330RF background, the transconjugant was affected in in vitro growth versus TX1330RF(pHylEfmTX16) and was attenuated in virulence; however, neither hylEfm nor hylEfm-down restored wild type function. We did not observe any in vivo effect on virulence of the other deletions of the hylEfm-region CONCLUSIONS: The four genes of the hylEfm region (including hylEfm) do not mediate the increased virulence conferred by pHylEfmTX16 in murine peritonitis. The use of the markerless counterselection system PheS* should facilitate the genetic manipulation of E. faecium in the future.

A functional collagen adhesin gene, acm, in clinical isolates of Enterococcus faecium correlates with the recent success of this emerging nosocomial pathogen.

Enterococcus faecium recently evolved from a generally avirulent commensal into a multidrug-resistant health care-associated pathogen causing difficult-to-treat infections, but little is known about the factors responsible for this change. We previously showed that some E. faecium strains express a cell wall-anchored collagen adhesin, Acm. Here we analyzed 90 E. faecium isolates (99% acm(+)) and found that the Acm protein was detected predominantly in clinically derived isolates, while the acm gene was present as a transposon-interrupted pseudogene in 12 of 47 isolates of nonclinical origin. A highly significant association between clinical (versus fecal or food) origin and collagen adherence (P

Distribution of genes encoding MSCRAMMs and Pili in clinical and natural populations of Enterococcus faecium.

Enterococcus faecium has recently emerged as an important cause of nosocomial infections. We previously identified 15 predicted surface proteins with characteristics of MSCRAMMs and/or pili and demonstrated that their genes were frequently present in 30 clinical E. faecium isolates studied; one of these, acm, has been studied in further detail. To determine the prevalence of the other 14 genes among various E. faecium populations, we have now assessed 433 E. faecium isolates, including 264 isolates from human clinical infections, 69 isolates from stools of hospitalized patients, 70 isolates from stools of community volunteers, and 30 isolates from animal-related sources. A variable distribution of the 14 genes was detected, with their presence ranging from 51% to 98% of isolates. While 81% of clinical isolates carried 13 or 14 of the 14 genes tested, none of the community group isolates and only 13% of animal isolates carried 13 or 14 genes. The presence of these genes was most frequent in endocarditis isolates, with 11 genes present in all isolates, followed by isolates from other clinical sources. The number of genes significantly associated with clinical versus fecal or animal origin (P = 0.04 to <0.0001) varied from 10 to 13, depending on whether comparisons were made against individual clinical subgroups (endocarditis, blood, and other clinical isolates) or against all clinical isolates combined as one group. The strong association of these genes with clinical isolates raises the possibility that their preservation/acquisition has favored the adaptation of E. faecium to nosocomial environments and/or patients.

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.