Adherence of Abiotrophia defectiva and Granulicatella species to fibronectin: is there a link with endovascular infections?

During a 6-year period, we isolated three Abiotrophia defectiva, six Granulicatella adiacens and two G. 'para-adiacens' strains from clinical specimens. All A. defectiva strains were isolated from immunocompetent patients with endovascular infections, whereas the Granulicatella spp. strains were isolated from immunosuppressed patients with primary bacteremia. As the capacity of bacteria to adhere to the host extracellular matrix (ECM) has been implicated in the pathogenesis of endovascular infection, we investigated the ability of A. defectiva and Granulicatella spp. isolates to bind different ECM components immobilized in microtiter plates. Adherence tests showed a strong attachment of A. defectiva strains to fibronectin, whereas Granulicatella spp. strains were not adherent. The poor adherence of Granulicatella spp. strains to the ECM could be correlated with a lower propensity to induce endocarditis.     

Serological properties of Abiotrophia and Granulicatella species (nutritionally variant streptococci)

Serological variations were examined among 12 type or reference strains and 91 oral isolates of vitamin B6-dependent Abiotrophia and Granulicatella spp. Rabbits were immunized with whole cells of 12 selected strains and 10 typing antisera were obtained, which were unreactive with the Lancefield group A to G antigen preparations. The reactivity of the antisera and autoclaved cell surface antigen extracts was tested by double diffusion in agar gel and a capillary precipitin test. These typing antisera categorized all Abiotrophia defectiva strains, all except one Granulicatella elegans strain, three-quarters of the Granulicatella adiacens, and half of the Granulicatella paraadiacens into 8 serotypes and 2 subserotypes. The Granulicatella balaenopterae type strain was unserotypable. All A. defectiva strains were serotype I, some of which were divided into subserotype I-1 and/or I-5. The G. adiacens strains generally belonged to serotype II or III, and the G. paraadiacens strains to serotype IV, V or VI. All G. adiacens or G. paraadiacens serotype II strains were also subserotype I-5. The G. elegans strains were serotype VII or VIII. These Abiotrophia and Granulicatella serotypes were undetectable among 33 strains of the other 11 species including the bacteriolytic enzyme-producing but vitamin B6-independent strains of Streptococcus, Enterococcus, Dolosigranulum and Aerococcus. The proposed serotyping system for Abiotrophia and Granulicatella spp. would be helpful in the identification and classification of these unique coccal isolates in ecological and epidemiological studies.     

Inhibition of fibronectin binding of some bacterial cells by subtle pH increase within the physiological range

The fibronectin (Fn)-binding ability of microorganisms is considered to be involved in their pathogenicities. Granulicatella adiacens, a member of the oral flora and a causative agent of culture-negative infective endocarditis, showed nearly maximum binding to immobilized Fn at pH 7.2 but greatly reduced binding at a slightly higher pH 7.4 and almost no binding at pH 7.6 in the presence of physiological concentration of NaCl (0.15 M). A similar pH-sensitive Fn-binding property was noted with Escherichia coli and Abiotrophia defectiva, but not with Streptococcus pyogenes nor Staphylococcus aureus. In contrast, bindings to laminin and fibrinogen observed for some of these strains were unaffected by the same pH changes. This fastidious pH-dependency of Fn-binding abilities of some bacteria warns that the pH condition must be seriously considered in the in vitro assay of bacterial adherence to fibronectin.     

Characterization of the bacteriolytic activity of nutritionally variant streptococci

The bacteriolytic activity of nutritionally variant streptococci (NVS), fastidious microaerophilic bacteria, which are members of the genera Abiotrophia and Granulicatella, was characterized in a renaturating SDS polyacrylamide gel electrophoresis system. Bacteriolytic profiles appeared quite different for the three species of NVS examined. Granulicatella adiacens or Abiotrophia defectiva each presented at least seven lytic bands, four of which were in common, while the other three were species-specific, whereas Granulicatella elegans showed six bands, which were overlapping with the G. adiacens bands. Four lytic bands were identified for enzymatic activity; D-alanyl-L-lysine hydrolase, endo-N-acetylglucosaminidase, endoacetylmuramidase, D-glutamyl-L-lysine hydrolase and acetylmuramoyl-L-alanine amidase activities could be defined. The bacteriolytic enzymes were purified and characterized for the kinetics of production during growth, autolytic activity, temperature and pH stability.     

Identification and characterization of bacterial-binding property in the type III repeat domain of fibronectin

To characterize fibronectin binding with Granulicatella adiacens, a causative agent of infective endocarditis, monoclonal antibodies were generated against human fibronectin and selected for their capacity to inhibit the fibronectin binding of the organism. Thermolysin and lysyl-endopeptidase digests of fibronectin were characterized by Western blot. The epitope of inhibitory monoclonal antibody was found in the central portion of fibronectin known as the cell-binding domain, and not in the N-terminal portion known to be the binding region of most microbial species, e.g., Staphylococcus aureus and Streptococcus pyogenes. While these two species could bind to both the N-terminal and central portion, Escherichia coli and G. adiacens bind only to the latter. Excess amounts of free fibronectin in the solution inhibited the bacterial adherence to the N-terminal fibronectin fragment, but not to the central region, thereby suggesting the central region plays a significant role for in vivo bacterial colonization in the presence of high concentrations of soluble fibronectin.     

Amoxycillin-resistant streptococci in dental plaque

This investigation was undertaken to study the prevalence of amoxycillin-resistant oral streptococci in normal healthy patients and patients with a cardiac condition, susceptible to infective endocarditis. Samples of supragingival dental plaque were collected from two test groups, children with congenital heart disease and adults with a history of rheumatic fever, and two control groups comprising normal healthy children and normal healthy adults. Bacteria from these samples were grown on a medium selective for oral streptococci, as well as on the same medium containing known concentrations of amoxycillin. The results indicate that a high percentage of rheumatic heart patients and children with congenital heart disease harboured amoxycillin-resistant oral streptococci. The level of amoxycillin resistance in the plaque of adults with rheumatic heart disease was significantly greater than in that of normal adults. In view of the high percentage of patients at risk harbouring amoxycillin-resistant streptococci, it is important that the individual clinical situation be monitored. Perhaps antibiotic sensitivity tests should be performed to select an appropriate antibiotic for prophylaxis of infective endocarditis.     

Screening of Probiotic Candidates in Human Oral Bacteria for the Prevention of Dental Disease

The oral cavity in healthy subjects has a well-balanced microbiota that consists of more than 700 species. However, a disturbance of this balance, with an increase of harmful microbes and a decrease of beneficial microbes, causes oral disorders such as periodontal disease or dental caries. Nowadays, probiotics are expected to confer oral health benefits by modulating the oral microbiota. This study screened new probiotic candidates with potential oral health benefits and no harmful effects on the oral cavity. We screened 14 lactobacillus strains and 36 streptococcus strains out of 896 oral isolates derived from healthy subjects. These bacteria did not produce volatile sulfur compounds or water-insoluble glucan, had higher antibacterial activity against periodontal bacteria, and had higher adherence activity to oral epithelial cells or salivary-coated hydroxyapatite in vitro. We then evaluated the risk of primary cariogenicity and infective endocarditis of the selected oral isolates. As a result, Lactobacillus crispatus YIT 12319, Lactobacillus fermentum YIT 12320, Lactobacillus gasseri YIT 12321, and Streptococcus mitis YIT 12322 were selected because they showed no cariogenic potential in an artificial mouth system and a lower risk of experimental infective endocarditis in a rat model. These candidates are expected as new probiotics with potential oral health benefits and no adverse effects on general health.     

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains.     

Binding of laminin to oral and endocarditis strains of viridans streptococci.

Attachment of bacteria to the host tissue is regarded as a crucial step in the development of many types of infections. Recent studies by us and others have shown that matrix proteins which serve as adhesion proteins for eucaryotic cells may also be recognized by some bacteria. In the present communication, we report that several strains of viridans streptococci are able to bind to laminin. Most strains isolated from blood and heart valves of patients with endocarditis expressed laminin receptors, whereas only a few of the strains isolated from the oral cavity recognized this protein. This observation indicates that laminin binding might be an important factor in the pathogenesis of viridans endocarditis. Laminin binding to two strains (Streptococcus mitis UAB594 and UAB597) isolated from patients with endocarditis was characterized further. The bacterial cells expressed a limited number of laminin receptors (4 X 10(2) to 1 X 10(3) per cell) which bound the protein in a high-affinity interaction (Kd, 40 to 80 nM). This receptor of S. mitis UAB594 was heat labile and could be solubilized from bacteria by brief digestion with trypsin. Solubilized receptors which competed with cell-bound receptors for 125I-laminin could be adsorbed on laminin-Sepharose but not on Sepharose substituted with fibrinogen or fibronectin. Comparison of laminin receptors from S. mitis with those previously described for Streptococcus pyogenes suggest that different sites in the laminin molecule are recognized by the two bacteria and hence that the corresponding receptor molecules are not identical.     

Interaction of clinical isolates of nonencapsulated Haemophilus influenzae with mammalian extracellular matrix proteins

The adherence of clinical isolates of nonencapsulated Haemophilus influenzae strains from patients with chronic bronchitis to distinct immobilized extracellular matrix components was determined. With selected strains the induction of plasmin formation by these isolates was studied. The strains could be divided into two groups: strains that showed a very high level of adherence to laminin and type I collagen, as well as adhesion to fibronectin and strains that showed only a moderate level of adhesion to laminin and a low level of adhesion to fibronectin. Plasmin formation was demonstrated for three out of eight isolates. Persisting and nonpersisting strains did not differ quantitatively or qualitatively with respect to the level of adhesiveness to the distinct matrix proteins and in their ability to induce plasmin formation.     

Hemicentins： What have we learned from worms？

Hemicentins are conserved extracellular matrix proteins discovered in Caenorhabditis elegans, with orthologs in all vertebrate species including human and mouse. Hemicentins share a single, highly conserved amino-terminal von Willebrand A domain, followed by a long （〉40） stretch of immunoglobulin repeats, multiple tandem epidermal growth factors and a fibulin-like carboxy-terminal module. C. elegans has a single hemicentin gene that has pleiotropic functions in transient cell contacts that are required for cell migration and basement membrane invasion and in stable contacts at hemidesmosome-mediated cell junctions and elastic fiber-like structures. Here, we summarize what is known about the function ofhemicentin in C. elegans and discuss implications for hemicentin function in other species.     

Amplification reaction to bacterial DNA for diagnosing infective endocarditis

The aim of this study was to evaluate the usefulness of broad-range bacterial PCR in infective endocarditis of bacterial etiology, and to determine its specificity and sensitivity. Twenty five blood samples were taken for analysis from patients with infective endocarditis and acquired valvular heart disease. Infective endocarditis was diagnosed according to Duke criteria. There were two control groups consisting of patients with acquired valvular heart disease: 10 patients with urinary tract infection and 15 patients without. Three different primer pairs for the region of the gene coding for 16S rRNA were tested, to find the most specific one. The highest specificity was found for F/R primers, as the relevant amplified PCR product was present in every blood sample with infective endocarditis, and also in 4 out of 10 patients with urinary tract infection. Broad-range PCR in bacterial endocarditis is a fast, sensitive and inexpensive tool for the detection of bacteria, but it is far more prone to contamination than species specific-PCR. However, in controlled conditions it may be valuable in the identification of non-specific infection allowing for a more rapid clinical diagnosis of endocarditis.     

Novel Tissue Level Effects of the Staphylococcus aureus Enterotoxin Gene Cluster Are Essential for Infective Endocarditis

BackgroundSuperantigens are indispensable virulence factors for Staphylococcus aureus in disease causation. Superantigens stimulate massive immune cell activation, leading to toxic shock syndrome (TSS) and contributing to other illnesses. However, superantigens differ in their capacities to induce body-wide effects. For many, their production, at least as tested in vitro, is not high enough to reach the circulation, or the proteins are not efficient in crossing epithelial and endothelial barriers, thus remaining within tissues or localized on mucosal surfaces where they exert only local effects. In this study, we address the role of TSS toxin-1 (TSST-1) and most importantly the enterotoxin gene cluster (egc) in infective endocarditis and sepsis, gaining insights into the body-wide versus local effects of superantigens.MethodsWe examined S. aureus TSST-1 gene (tstH) and egc deletion strains in the rabbit model of infective endocarditis and sepsis. Importantly, we also assessed the ability of commercial human intravenous immunoglobulin (IVIG) plus vancomycin to alter the course of infective endocarditis and sepsis.ResultsTSST-1 contributed to infective endocarditis vegetations and lethal sepsis, while superantigens of the egc, a cluster with uncharacterized functions in S. aureus infections, promoted vegetation formation in infective endocarditis. IVIG plus vancomycin prevented lethality and stroke development in infective endocarditis and sepsis.ConclusionsOur studies support the local tissue effects of egc superantigens for establishment and progression of infective endocarditis providing evidence for their role in life-threatening illnesses. In contrast, TSST-1 contributes to both infective endocarditis and lethal sepsis. IVIG may be a useful adjunct therapy for infective endocarditis and sepsis.     

Streptococcus Adherence and Colonization

Summary: Streptococci readily colonize mucosal tissues in the nasopharynx; the respiratory, gastrointestinal, and genitourinary tracts; and the skin. Each ecological niche presents a series of challenges to successful colonization with which streptococci have to contend. Some species exist in equilibrium with their host, neither stimulating nor submitting to immune defenses mounted against them. Most are either opportunistic or true pathogens responsible for diseases such as pharyngitis, tooth decay, necrotizing fasciitis, infective endocarditis, and meningitis. Part of the success of streptococci as colonizers is attributable to the spectrum of proteins expressed on their surfaces. Adhesins enable interactions with salivary, serum, and extracellular matrix components; host cells; and other microbes. This is the essential first step to colonization, the development of complex communities, and possible invasion of host tissues. The majority of streptococcal adhesins are anchored to the cell wall via a C-terminal LPxTz motif. Other proteins may be surface anchored through N-terminal lipid modifications, while the mechanism of cell wall associations for others remains unclear. Collectively, these surface-bound proteins provide Streptococcus species with a “coat of many colors,” enabling multiple intimate contacts and interplays between the bacterial cell and the host. In vitro and in vivo studies have demonstrated direct roles for many streptococcal adhesins as colonization or virulence factors, making them attractive targets for therapeutic and preventive strategies against streptococcal infections. There is, therefore, much focus on applying increasingly advanced molecular techniques to determine the precise structures and functions of these proteins, and their regulatory pathways, so that more targeted approaches can be developed.     

Extracellular ABTS-oxidizing activity of autochthonous fungal strains from Argentina in solid medium

The screening for extracellular oxidases and peroxidases from autochthonous filamentous fungi isolated from different substrates is an important step towards the detection of extracellular fungal oxidative systems. Thirty-one autochthonous fungal strains from Argentina, belonging to different ecophysiological and taxonomic groups, were plate-screened for their ability to produce extracellular oxidoreductases. Modified Kirk solid medium containing the chromogen 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) was used to determine the presence of this extracellular activity. The fungi tested were grouped according to the colour intensity of the modified Kirk medium in: a) species without extracellular ABTS-oxidizing activity; b) species with low extracellular ABTS-oxidizing activity; c) species with moderate extracellular ABTS-oxidizing activity; d) species with high extracellular ABTS-oxidizing activity. The assay revealed extracellular ABTS-oxidizing activity in 90% of the strains tested. All species of Basidiomycetes used exhibited ABTS-oxidizing activity, except Laeticorticium roseum. Aspergillus terreus and Epicoccum purpurascens (Deuteromycetes) did not show extracellular oxidative activity on ABTS. Agrocybe aegerita, Amauroderma boleticeum, Cladosporium cladosporioides, Coriolopsis rigida, Grammothele subargentea, Graphium putredinis, Hexagona hydnoides, Hexagona papyraceae, Loweporus lividus, Peniophora albobadia, Phellinus everhartii, Phellinus gilvus; Phellinus linteus; Pleurotus laciniatocrenatus, Pycnoporus sanguineus, Rigidoporus ulmarius, Steccherinum rawakense, Talaromyces helicus, Trametes elegans, Trametes pavonia, Trametes villosa and Trichaptum sector are reported here for the first time as species capable of producing ABTS-oxidizing extracellular oxidorreductases.     

Oral Streptococci Utilize a Siglec-Like Domain of Serine-Rich Repeat Adhesins to Preferentially Target Platelet Sialoglycans in Human Blood

Damaged cardiac valves attract blood-borne bacteria, and infective endocarditis is often caused by viridans group streptococci. While such bacteria use multiple adhesins to maintain their normal oral commensal state, recognition of platelet sialoglycans provides an intermediary for binding to damaged valvular endocardium. We use a customized sialoglycan microarray to explore the varied binding properties of phylogenetically related serine-rich repeat adhesins, the GspB, Hsa, and SrpA homologs from Streptococcus gordonii and Streptococcus sanguinis species, which belong to a highly conserved family of glycoproteins that contribute to virulence for a broad range of Gram-positive pathogens. Binding profiles of recombinant soluble homologs containing novel sialic acid-recognizing Siglec-like domains correlate well with binding of corresponding whole bacteria to arrays. These bacteria show multiple modes of glycan, protein, or divalent cation-dependent binding to synthetic glycoconjugates and isolated glycoproteins in vitro. However, endogenous asialoglycan-recognizing clearance receptors are known to ensure that only fully sialylated glycans dominate in the endovascular system, wherein we find these particular streptococci become primarily dependent on their Siglec-like adhesins for glycan-mediated recognition events. Remarkably, despite an excess of alternate sialoglycan ligands in cellular and soluble blood components, these adhesins selectively target intact bacteria to sialylated ligands on platelets, within human whole blood. These preferred interactions are inhibited by corresponding recombinant soluble adhesins, which also preferentially recognize platelets. Our data indicate that circulating platelets may act as inadvertent Trojan horse carriers of oral streptococci to the site of damaged endocardium, and provide an explanation why it is that among innumerable microbes that gain occasional access to the bloodstream, certain viridans group streptococci have a selective advantage in colonizing damaged cardiac valves and cause infective endocarditis.     

Identification of Actinomyces,Propionibacteria, Lactobacilli and Bifidobacteria by Amplified 16S rDNA Restriction Analysis

Amplified 16S rDNA restriction analysis (ARDRA) with Hae III and Hpa II was applied to 37 reference strains, 179 human clinical and four veterinary isolates of Propionibacterium, Lactobacillus andBifidobacterium and some other anaerobic, non-sporing, Gram-positive bacilli. Results were compared with those obtained by ARDRA for reference strains (26) and clinical isolates (469) of Actinomyces spp. Reference strains were clearly differentiated to species level. Clinical isolates of Propionibacterium and Lactobacillus were identified with confidence to species level. Bifidobacterium spp. were identified in ARDRA with confidence to genus, but anomalies in species level identification of some reference strains and clinical isolates may reflect unreliable identification in conventional tests. Isolates of Arcanobacterium spp., Actinobaculum schaalii, Eggerthella lenta, some Eubacterium spp., Gardnerella vaginalis, Mobiluncus spp., Atopobium vaginae, Abiotrophia defectiva, Streptococcus mutans, Streptococcus intermedius and Clostridium sp. were clearly differentiated in ARDRA. ARDRA is a simple, rapid, and highly discriminatory method for identification of anaerobic, non-sporing, Gram-positive bacilli.     

Papilin, a novel component of basement membranes, in relation to ADAMTS metalloproteases and ECM development

Papilins are homologous, secreted extracellular matrix proteins which share a common order of protein domains. They occur widely, from nematodes to man, and can differ in the number of repeats of a given type of domain. Within one species the number of repeats can vary by differential RNA splicing. A distinctly conserved cassette of domains at the amino-end of papilins is homologous with a cassette of protein domains at the carboxyl-end of the ADAMTS subgroup of secreted, matrix-associated metalloproteases. Papilins primarily occur in basement membranes. Papilins interact with several extracellular matrix components and ADAMTS enzymes. Papilins are essential for embryonic development of Drosophila melanogaster and Caenorhabditis elegans.     

Propionibacterium acnes in the etiology of endocarditis]

Propionibacterium acnes is the gram positive anaerobic bacteria belongs to the normal skin and oral microbial flora. The participation of this microorganism in the infective endocarditis is still controversial. The aim of the study was to perform the diagnostic and therapeutic difficulties in 5 patients with infective endocarditis caused by Propionibacterium acnes. In 3 out of 5 patients the infective endocarditis developed after prosthesis valve replacement, in 2 others on the native valves. The inserted prostheses were mechanical ones, propionibacterium acnes was identified as causative organisms in all of the causes (two positive blood and/or valve culture). The bacterial strains were sensitive to the antibiotics as: penicillins, cephalosporins, clindamycin, and vancomycin, however cephalosporins used at the beginning of the treatment in 3 patients and clindamycin in 1 patient had limited clinical efficacy. Later treatment with timentin, augmentin and tienamycin was successful in 3 patients; one patient was cured with vancomycin. One patient died because of septic, embolic complication in early stage of illness. We conclude the effectiveness of penicillins in combination with clavulanic acid and tienamycin in therapy of infective endocarditis due to Propionibacterium acnes. The treatment should be lasted during 4-6 weeks.     

Platelet receptor polymorphisms do not influence Staphylococcus aureus-platelet interactions or infective endocarditis

Cardiac vegetations result from bacterium-platelet adherence, activation and aggregation, and are associated with increased morbidity and mortality in infective endocarditis. The GPIIb/IIIa and FcγRIIa platelet receptors play a central role in platelet adhesion, activation and aggregation induced by endocarditis pathogens such as Staphylococcus aureus, but the influence of known polymorphisms of these receptors on the pathogenesis of infective endocarditis is unknown. We determined the GPIIIa platelet antigen Pl(A1/A2) and FcγRIIa H131R genotype of healthy volunteers (n?=?160) and patients with infective endocarditis (n?=?40), and investigated the influence of these polymorphisms on clinical outcome in infective endocarditis and S. aureus-platelet interactions in vitro. Platelet receptor genotype did not correlate with development of infective endocarditis, vegetation characteristics on echocardiogram or the composite clinical end-point of embolism, heart failure, need for surgery or mortality (P?>?0.05 for all), even though patients with the GPIIIa Pl(A1/A1) genotype had increased in vivo platelet activation (P?=?0.001). Furthermore, neither GPIIIa Pl(A1/A2) nor FcγRIIa H131R genotype influenced S. aureus-induced platelet adhesion, activation or aggregation in vitro (P?>?0.05). Taken together, our data suggest that the GPIIIa and FcγRIIa platelet receptor polymorphisms do not influence S. aureus-platelet interactions in vitro or the clinical course of infective endocarditis.