In-vitro stimulation of TNF-alpha from human whole blood by cell-free supernatants of gram-positive bacteria.

Gram-positive bacteria are being recognized increasingly as the cause of shock-like syndromes, clinically indistinguishable from those seen in association with Gram-negative endotoxic shock. Much clinical and experimental data link tumour necrosis factor-alpha (TNF-alpha) with the pathogenesis of endotoxic shock, and a number of studies of individual Gram-positive species have also implicated TNF-alpha. We report here the first systematic study of the ability of cell-free supernatants of common Gram-positive bacteria to induce TNF-alpha from human peripheral blood monocytes in vitro. Almost all the 63 strains were able to induce TNF-alpha, although the levels were substantially lower than those obtained from supernatants of Gram-negative bacteria, used as controls. Streptococcus pneumoniae, S. pyogenes, viridans streptococci and coagulase-negative staphylococci were consistently more active than group B and D streptococci. TNF-alpha induction did not correlate with conventional markers of pathogenicity; amongst strains of Staphylococcus aureus, commensal and blood culture isolates did not induce significantly different amounts of TNF. We conclude that cell-free supernatants of most Gram-positive bacteria are capable of inducing TNF-alpha from human peripheral blood monocytes in vitro, but the significance of this finding remains to be determined.     

Beta 2 integrins are involved in cytokine responses to whole Gram-positive bacteria

Proinflammatory cytokines have an important pathophysiologic role in septic shock. CD14 is involved in cytokine responses to a number of purified bacterial products, including LPS. However, little is known of monocyte receptors involved in cytokine responses to whole bacteria. To identify these receptors, human monocytes were pretreated with different mAbs and TNF-alpha was measured in culture supernatants after stimulation with whole heat-killed bacteria. Human serum and anti-CD14 Abs significantly increased and decreased, respectively, TNF-alpha responses to the Gram-negative Escherichia coli. However, neither treatment influenced responses to any of the Gram-positive bacteria tested, including group A and B streptococci, Listeria monocytogenes, and Staphylococcus aureus. Complement receptor type III (CR3 or CD18/CD11b) Abs prevented TNF-alpha release induced by heat-killed group A or B streptococci. In contrast, the same Abs had no effects when monocytes were stimulated with L. monocytogenes or S. aureus. Using either of the latter bacteria, significant inhibition of TNF-alpha release was produced by Abs to CD11c, one of the subunits of CR4. To confirm these blocking Ab data, IL-6 release was measured in CR3-, CR4-, or CD14-transfected Chinese hamster ovary cells after bacterial stimulation. Accordingly, streptococci triggered moderate IL-6 production (p < 0.05) in CR3 but not CD14 or CR4 transfectants. In contrast, L. monocytogenes and S. aureus induced IL-6 release in CR4 but not CR3 or CD14 transfectants. Collectively our data indicate that beta 2 integrins, such as CR3 and CR4, may be involved in cytokine responses to Gram-positive bacteria. Moreover, CD14 may play a more important role in responses to whole Gram-negative bacteria relative to Gram-positive ones.     

Quorum sensing in streptococcal biofilm formation

Bacteria in their natural ecosystems preferentially grow as polysaccharide-encased biofilms attached to surfaces. Although quorum-sensing (QS) systems directing the 'biofilm phenotype' have been extensively described in Gram-negative bacteria, there is little understanding of the importance of these systems in Gram-positive biofilm formation. Streptococci are a diverse group of Gram-positive bacteria that colonize epithelial, mucosal and tooth surfaces of humans. In several streptococci, competence-stimulating peptide (CSP)-mediated QS has been connected with competence development for genetic transformation. Recent work, especially with bacteria that inhabit the biofilm of dental plaque, has linked CSP stimuli to other cell-density adaptations, such as biofilm formation.     

Rapid labelling of bacteria with [14C]palmitic acid and its application in an aggregation assay

Abstract A rapid and simple procedure for labelling bacterial cells based on binding of [¹⁴C]palmitic acid to the bacterial surface is described. The method was found convenient for both Gram-positive and Gram-negative bacteria such as staphylococci, streptococci and Salmonella . Some factors affecting the binding of [¹⁴C]palmitic acid to the surface of streptococci were examined. Treatment of bacteria with heat, trypsin or β-galactosidase had no effect on labelling. The binding was relatively independent of ionic strength in the range 0.1–1 M NaCl, but was dependent on pH and presence of detergents. The [¹⁴C]palmitic acid labelling method was tested in studies of aggregation of oral streptococci. The aggregation assay was sensitive and very reproducible.     

Biofilm mode of growth of Streptococcus intermedius favored by a competence-stimulating signaling peptide

Gram-positive and gram-negative bacteria use quorum sensing to coordinate population behavior. In several streptococci, quorum sensing mediated by competence-stimulating peptides (CSP) is associated with development of competence for transformation. We show here that a synthetic CSP favored the biofilm mode of growth of Streptococcus intermedius without affecting the rate of culture growth.     

Avoparcin and vancomycin: useful antibiotics for the isolation of brewery lactic acid bacteria

Vancomycin (30 μg) and avoparcin (20 μg) have no effect on growth of hetero-fermentative lactobacilli, mesophilic homofermentative lactobacilli, Lactobacillus salivarius and bacteria of the genera Leuconostoc and Pediococcus. The growth of thermophilic homofermentative lactobacilli (with the exception of Lacto salivarius ), enteric and lactic streptococci and all other Gram-positive bacteria encountered in breweries (and likely to be encountered in foods) is inhibited by these antibiotics. The recovery of sub-lethally stressed cells is slightly reduced by the presence of either antibiotic. Vancomycin and avoparcin are useful selective agents for the detection of lactic acid bacteria in the brewing industry and may also be of use, for quality control purposes, in the distilling, wine, cider, dairy and meat industries. These antibiotics may also find applications in differentiation of pediococci from streptococci, and in rapid identification of lactobacilli.     

Characterization of aid1, a Novel Gene Involved in Fusobacterium nucleatum Interspecies Interactions

The oral opportunistic pathogen Fusobacterium nucleatum is known to interact with a large number of different bacterial species residing in the oral cavity. It adheres to a variety of Gram-positive bacteria, including oral streptococci via the arginine-inhibitable adhesin RadD. In this study, we describe a novel protein encoded by the predicted open reading frame FN1253 that appears to play a role in interspecies interactions of F. nucleatum, particularly with oral streptococci and related Gram-positive species. We designated FN1253 as aid1 (Adherence Inducing Determinant 1). Expression analyses demonstrated that this gene was induced in F. nucleatum single species biofilms, while the presence of representative members of the oral microbiota known to adhere to F. nucleatum triggered its suppression. Inactivation as well as overexpression of aid1 affected the ability of F. nucleatum to coaggregate with oral streptococci and the closely related Enterococcus faecalis, but not other Gram-positive oral species tested. Furthermore, overexpression of aid1 led to a drastic change in the structure of dual species biofilms of F. nucleatum with oral streptococci. Aid1 function was abolished in the presence of arginine and found to be dependent on RadD. Interestingly, differential expression of aid1 did not affect messenger RNA and protein levels of RadD. These findings indicate that RadD-mediated adhesion to oral streptococci involves more complex cellular processes than the simple interaction of adhesins on the surface of partner strains. Aid1 could potentially play an important role in facilitating RadD-mediated interaction with oral streptococci by increasing binding specificity of F. nucleatum to other microbial species.     

Selectivity of Mitis Salivarius agar and a new selective medium for oral streptococci in dogs

An evaluation on the applicability of Mitis Salivarius agar (MS) medium, commonly used for the detection of oral streptococci in human and animals, to dog specimens and the development of a new selective medium for isolating streptococci from the canine oral cavity are described. Oral samples from dogs were cultured on MS medium under anaerobic conditions. The predominant facultative anaerobic bacteria on MS plates were gram-negative rods. Selectivity of streptococci on MS medium was 21.2%. A new selective medium, designated MS-CAN-AE, was developed for the isolation of streptococci from the canine oral cavity. The average growth recovery of laboratory and clinically isolated strains of streptococci on MS-CAN-AE medium was 84.1% of that on MS medium. Gram-positive rods and gram-negative rods and cocci rarely grew on the MS-CAN-AE. The selectivity of MS-CAN-AE was 95.0% for clinical samples. MS-CAN-AE medium will be helpful for investigations of streptococci in the canine oral cavity.     

Microflora of the posterior pharyngeal wall, larynx and postoperative wounds in patients after laryngectomy]

The study group were persons with risk factors of colonization by pathogenic strains and included smokers, patients suffering from paradontosis, and patients with visibly neglected oral cavity and teeth. We isolated and classified to the species or genera 488 microorganisms. Of posterior pharyngeal wall flora, 61% of were Gram-negative bacteria, represented predominantly by Haemophilus and anaerobic rods and aerobic cocci belonging to the Neisseria and Moraxella (Branhamella) genera. In the group of Gram-positive cocci (34% of the total number of microorganisms), oral streptococci, Stomatococcus mucilaginosus and Streptococcus pneumoniae were isolated most frequently. The affected by neoplastic lesions larynx, was colonized by similar bacterial groups. However, the incidence of Gram-positive cocci was higher. The main etiologic factor of purulent post-operative wound inflammations were methicillin-resistant Staphylococcus aureus strains (MRSA), which had been absent among the bacteria isolated from patients on admission.     

Bacteriological activity in unfiltered calf sera collected for tissue culture use.

Bacteriological tests were made on 24 lots of unfiltered calf serum collected for subsequent use as a component of tissue culture media. The examination included the isolation and identification of bacteria, assay of phages, and demonstration of endotoxin material. Only Gram-positive bacteria were isolated and 96% of the sera were contaminated with bacteria. The prevalent strains of bacteria found were Bacillus species and streptococci and 63% of the sera coagulated Limulus amebocyte lysate. More than 90% of the lots contained phages demonstrable with the C-3000 strain of Escherichia coli. Only one lot of the serum was found to be free from bacteria, phages, and endotoxin by the tests used.     

Immunological crossreactivity to the catabolite control protein CcpA from Bacillus megaterium is found in many Gram-positive bacteria

Abstract The catabolite control protein CcpA from Bacillus megaterium was overproduced as a fusion protein to a 6xhis affinity tag and purified to homogeneity. Polyclonal antibodies of high affinity and specificity were raised against the purified protein. The serum did not crossreact with purified Lac repressor despite the fact that CcpA and LacI belong to the same protein family. Using this antiserum we identified proteins that share antigenic determinants with CcpA in many Gram-positive bacteria, including bacilli, staphylococci, streptococci, lactic acid bacteria, and some actinomycetes.     

Platelet and neutrophil responses to Gram positive pathogens in patients with bacteremic infection

Background

Many Gram-positive pathogens aggregate and activate platelets in vitro and this has been proposed to contribute to virulence. Platelets can also form complexes with neutrophils but little is however known about platelet and platelet-neutrophil responses in bacterial infection.

Methodology/Principal Findings

We added isolates of Gram-positive bacteria from 38 patients with a bacteremic infection to blood drawn from the same patient. Aggregometry and flow cytometry were used to assess platelet aggregation and to quantify activation of platelets, neutrophils, and platelet-neutrophils complexes (PNCs) induced by the bacteria. Fifteen healthy persons served as controls. Most isolates of Staphylococcus aureus, beta hemolytic streptococci, and Enterococcus faecalis induced aggregation of platelets from their respective hosts, whereas pneumococci failed to do so. S. aureus isolates induced platelet aggregation more rapidly in patients than in controls, whereas platelet activation by S. aureus was lower in patients than in controls. PNCs were more abundant in baseline samples from patients than in healthy controls and most bacterial isolates induced additional PNC formation and neutrophil activation.

Conclusion/Significance

We have demonstrated for the first time that bacteria isolated from patients with Gram-positive bacteremia can induce platelet activation and aggregation, PNC formation, and neutrophil activation in the same infected host. This underlines the significance of these interactions during infection, which could be a target for future therapies in sepsis.     

Bacteriological activity in unfiltered calf sera collected for tissue culture use

Summary Bacteriological tests were made on 24 lots of unfiltered calf serum collected for subsequent use as a component of tissue culture media. The examination included the isolation and identification of bacteria, assay of phages, and demonstration of endotoxin material. Only Gram-positive bacteria were isolated and 96% of the sera were contaminated with bacteria. The prevalent strains of bacteria found wereBacillus species and streptococci and 63% of the sera coagulatedLimulus amebocyte lysate. More than 90% of the lots contained phages demonstrable with the C-3000 strain ofEscherichia coli. Only one lot of the serum was found to be free from bacteria, phages, and endotoxin by the tests used.     

Molecular taxonomy and phylogenetic position of lactic acid bacteria

Lactic acid bacteria, important in food technology, are Gram-positive organisms exhibiting a DNA G + C content of less than 50 mol%. Phylogenetically they are members of the Clostridium-Bacillus subdivision of Gram-positive eubacteria. Lactobacillus and streptococci together with related facultatively anaerobic taxa evolved as individual lines of descent about 1.5-2 billion years ago when the earth passed from an anaerobic to an aerobic environment. In contrast to the traditional, morphology-based classification, the genus Lactobacillus is intermixed with strains of Pediococcus and Leuconostoc. Similarly, the physiology-based clustering of lactobacilli into Thermo-, Strepto- and Betabacterium does not agree with their phylogenetic relationships. On the other hand, the phenotypically defined genus Streptococcus is not a phylogenetic coherent genus but its members fall into at least 3 moderately related genera, i.e. Streptococcus, Lactococcus and Enterococcus. The genus Bifidobacterium, frequently grouped with the lactobacilli, is the most ancient group of the second, the Actinomycetes subdivision of the Gram-positive eubacteria. In addition, propionibacteria, microbacteria and brevibacteria belong to this subdivision but the latter organisms appear as offshoots of non-lactic acid bacteria.     

α-溶血性链球菌在健康人群口咽部分布及 生物拮抗作用

目的通过采集健康人群口咽部分泌物,分析上呼吸道中α-溶血性链球菌的分布状况,并对革兰阳性化脓性球菌进行生物拮抗试验,为进一步研究上呼吸道益生菌提供理论基础。方法随机自愿原则,用无菌咽拭子采集沈阳市年龄在3～75岁的300名健康人群咽后壁分泌物,对α-溶血性链球菌进行鉴定和定量分析。对致病菌的生物拮抗试验采用小缸杯法。结果定量分析显示不同年龄人群咽后壁的α-溶血性链球菌检出率均较高。在咽后壁菌群中α-溶血性链球菌构成比最多的是幼儿组,达到60.3%。其中唾液链球菌群在幼儿组所占比重最大;老年组人群格氏链球菌占比较大;儿童、青年、成人以缓症链球菌和口腔链球菌为主。对革兰阳性化脓性球菌的生物拮抗试验显示,1株婴儿链球菌婴儿亚种能够拮抗8株致病菌;4株分离菌只能拮抗1株病原菌,提示不同的菌株拮抗病原菌的能力差异较大。结论α-溶血性链球菌在人群中分布广,数量多,不同年龄人群的菌群构成存在差异。并且某些菌株显示出对致病菌较强的生物拮抗作用,推测这些菌株在呼吸道黏膜保护中起到重要作用,可作为上呼吸道益生菌的备选菌株。     

Anaerobic microbiology in 198 cases of pleural empyema: a Bulgarian study

The aim of the study was to evaluate the incidence of anaerobic bacteria in 198 patients with pleural empyema and the susceptibility of isolates to eight antibacterial agents. Isolates were identified by the Crystal anaerobes identification system, API System rapid ID 32 A and/or routine methods. Susceptibility was tested by Sceptor MIC system for anaerobic bacteria and limited agar dilution method. Anaerobic bacteria were found in 74.2% of the patients and included 247 strains within 21 genera. The predominant anaerobes were Gram-positive anaerobic cocci (52 isolates), Fusobacterium (51), microaerophilic streptococci (24), Prevotella (19) and Bacteroides species (11). Common species/groups were Fusobacterium nucleatum (in 27.2% of specimens yielding anaerobes), Micromonas micros (8.2%), Finegoldia magna (7.5%), Bacteroides fragilis group (6.8%), Peptostreptococcus anaerobius (6.1%) and F. necrophorum (5.4%). No resistance to chloramphenicol and ampicillin/sulbactam was detected. The susceptibility rates of Gram-negative anaerobic isolates to penicillin, cefoxitin, clindamycin, clarithromycin, metronidazole and tetracycline were 63.8%, 90.2%, 87.8%, 58.6%, 98.8% and 71%, and those of Gram-positive anaerobes were 79.2%, 100%, 84.3%, 68.4%, 41.9% and 75%, respectively. The wide diversity of isolated anaerobic genera and species and the susceptibility patterns of the isolates emphasize the role of the anaerobic microbiology in cases of pleural empyema.     

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.