Genetic characterization of the lobster pathogen Aerococcus viridans var. homari by 16S rRNA gene sequence and RAPD

A combination of 16S rRNA sequencing and random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity within Aerococcus viridans var. homari, the causative agent of gaffkemia in lobsters. A collection of 7 A. viridans var. homari strains and 2 avirulent A. viridans-like cocci isolated from homarid lobsters harvested from different regions on the Atlantic Coast of North America were analyzed. The isolates are separated geographically and temporally between the years 1947 and 2000. Sequencing of 16S rRNA genes confirmed the inclusion of all 9 isolates in the monophyletic A. viridans clade (99.8 to 100% similarity). RAPD analysis revealed that the 9 A. viridans var. homari isolates could be separated into 2 distinct subtypes. Subtype 1 included the 7 pathogenic lobster isolates and constituted a homogeneous group regardless of their geographical, temporal or virulence differences. Subtype 2 contained the 2 avirulent A. viridans-like cocci that had distinct RAPD patterns and clustered separately with the non-marine A. viridans. RAPD analysis represented a useful method for determining molecular subtyping for the intraspecific classification and epidemiological investigations of A. viridans var. homari.     

Broad-host-range shuttle vectors for screening of regulated promoter activity in viridans group streptococci: isolation of a pH-regulated promoter

Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the hydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.     

Broad-Host-Range Shuttle Vectors for Screening of Regulated Promoter Activity in Viridans Group Streptococci: Isolation of a pH-Regulated Promoter

Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the hydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.     

Use of dye affinity chromatography for the purification of Aerococcus viridans lactate oxidase

Lactate oxidase was purified from Aerococcus viridans (A. viridans) by dye affinity chromatography and FPLC ion exchange chromatography. The lactate oxidase could be purified by comparatively simple procedures, the purification achieved from a crude extract of A. viridans was 41-fold with a specific activity of 143 units/(mg of protein). The purified enzyme was a L-lactate oxidase, which catalyses the conversion of L-lactate in the presence of molecular oxygen to pyruvate and H(2)O(2). This purified lactate oxidase showed an apparent molecular mass of 48,200 in SDS-PAGE and the native molecular weight, as estimated by FPLC gel filtration, was 187,300. This molecular weight indicates that lactate oxidase exists in tetrameric form after gel filtration. To differing degrees, all the triazine dyes tested were inhibitors of lactate oxidase, solutions of free triazine dyes showing an inhibition mechanism which was both time- and pH-dependent.     

The direct application of the polymerase chain reaction to DNA extracted from foods

Two methods for the successful extraction of DNA from foods are described. The rapid lysis method uses a proteinase K buffer system to lyse cells and solubilize food samples. DNA is then precipitated using isopropanol. The second method achieves cell lysis using toluene and mutanolysin, and solubilization using guanidium thiocyanate. Following protein removal with organic solvents DNA is precipitated with isopropanol. Both methods enabled the polymerase chain reaction to be applied directly to DNA extracted from samples of cheese, coleslaw and raw chicken and allowed the direct rapid, sensitive and specific detection of Yersinia enterocolitica, Aerococcus viridans and Listeria monocytogenes in these foods.     

Presumptive Identification of Group A, B, and D Streptococci

A battery of five tests was used for presumptive identification of the pathogenic streptococci. The non-serological methods included determination of hemolysis for all strains, bacitracin susceptibility for group A streptococci, hippurate hydrolysis by group B streptococci, and bile-esculin reaction for group D streptococci. Enterococcal group D streptococci were differentiated from non-enterococcal group D streptococci by 6.5% NaCl tolerance. Two other categories of streptococci resulted: beta-hemolytic streptococci non-groups A, B, or D; and alpha- or nonhemolytic streptococci, not enterococci, not further identified (viridans streptococci). The tests were used as a battery and not as single entities. In this manner more than 99% of the group A, 99% of the group B, 81% of the beta-hemolytic streptococci non-group A, B, or D, 99% of the group D enterococci, 97% of the group D non-enterococci, and 94% of the viridans streptococci were correctly identified.     

Septicaemia associated with an Aerococcus viridans infection in immunodeficient mice

This report describes a case series of septicaemia caused by infection with Aerococcus viridans in immunodeficient NOD/LtSz-Prkdc(scid) (NOD/SCID) mice. During a period of 3 weeks more than 40 animals died or became ill with clinical signs of ruffled coat, weight loss, laboured breeding, and distended abdomen. At necropsy it was found that the animals displayed symptoms of sepsis with widespread abscesses in the liver, heart, lungs or pyogenic peritonitis. A Gram-positive coccus was isolated in pure culture from the abscesses or peritoneum from affected animals. According to phenotypic and phylogenetic characterization, the isolate was identified as A. viridans. This is the first report of a spontaneous outbreak of septicaemia caused by A. viridans infection in immunodeficient laboratory mice and we conclude that A. viridans should be considered as a pathogen in immunodeficient mice.     

Highly efficient Aerococcus viridans L-α-glycerophosphate oxidase production in the presence of H2O2-decomposing agent: purification and kinetic characterization

Glycerophosphate oxidase was purified from Aerococcus viridans cells by phase partitioning in Triton X-114, ammonium sulfate fractionation, FPLC ion-exchange chromatography and FPLC hydrophobic-interaction chromatography. The purification achieved from a crude extract of A. viridans was 38-fold with a 32% recovery of activity. Under the growth conditions used, A. viridans strain CECT 978 proved to be an excellent glycerophosphate-oxidase producer, with enzyme production 2,800-fold greater than that described in the literature for the same microorganism. The culture medium used in the present work is that commonly used for cultivation of this microorganism, except that an H2O2-decomposing enzyme was added. The addition of catalase to the growth medium had a clear effect on the growth rate. Furthermore, methylglyoxal, a metabolite that is formed enzymatically from triose phosphates, was found to be an inactivator of glycerophosphate oxidase activity.     

Esophageal diverticulum associated with Aerococcus viridans infection in a loggerhead sea turtle (Caretta caretta)

A juvenile loggerhead sea turtle (Caretta caretta) stranded in Gran Canaria, Spain was necropsied. The turtle was underweight, had sunken eyes, and small amounts of crude oil were in the oral cavity. The most significant lesion was a large esophageal diverticulum at the junction of the esophagus and stomach. The diverticulum was full of gas and green mucoid fluid and a diffuse thick yellow fibrinonecrotic membrane covered the mucosa. The lumen of the diverticulum also contained moderate numbers of cephalopods, crustaceans, and anthropogenic debris including crude oil balls, plastics, and fishing lines. Histologically there was a severe diffuse fibrinonecrotic esophagitis. Aerococcus viridans was isolated from the diverticulum. This is the first report of an esophageal diverticulum in a sea turtle. Although A. viridans is a known pathogen of lobsters and fishes, there are no reports of A. viridans infection in sea turtles.     

Co-immobilization of lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase onto alkylamine glass beads through glutaraldehyde coupling

A method for co-immobilizing lipase from porcine pancreas, glycerol kinase (GK) from Cellulomonas spp., glycerol-3-phosphate oxidase (GPO) from Aerococcus viridans and peroxidase from horseradish onto zirconia-coated alkylamine glass beads through glutaraldehyde coupling has been described. The co-immobilized enzymes retained 71.4% of initial specific activity with a conjugation yield of 43.6 mg/g support. The optimum pH and Km for triolein increased, while Vmax was decreased slightly, but incubation temperature for maximum activity remained unaltered after co-immobilization. The co-immobilized enzymes showed increased thermal and storage stabilities in cold, compared to their native form. Among the various metal salts tested, only CuSO4 caused inhibition of both free and co-immobilized enzymes. The co-immobilized enzymes showed better suitability over mixture of individually immobilized enzymes in determination of serum triglyceride.     

Aerococcus viridans in the Hospital Environment

Aerococcus viridans has been described as an airborne organism prevalent in occupied rooms. It has also been described as an organism having many characteristics that might cause it to be confused with streptococci or staphylococci, and this may account for the fact that the presence of A. viridans has not been reported in the hospital environment or in clinical specimens. Swab specimens were taken from 47 objects in 11 different areas in a local hospital, cultured overnight in Trypticase Soy Broth, and streaked on blood-agar and on a selective serum agar containing potassium tellurite and crystal violet. Of 85 alpha-hemolytic cultures isolated, 11 proved to be typical A. viridans based on diagnostic tests that also were applied to a collection of gram-positive cocci, including authentic strains of A. viridans. These organisms are gram-positive cocci with a strong tendency toward tetrad formation in broth cultures. They are predominantly aerobic, have a very weak catalase activity, and lack porphyrin respiratory enzymes. Three similar cultures also were obtained from routine clinical specimens.     

Genome sequence of Aerococcus viridans LL1

Aerococcus viridans is a catalase-negative Gram-positive bacterium and has been described as an airborne organism widely distributed in the hospital environment or in clinical specimens. We isolated A. viridans strain LL1 from indoor dust samples collected by a patient. Here, we prepared a genome sequence for this strain consisting of 31 contigs totaling 1,994,039 bases and a GC content of 39.42%.     

Covalently immobilized DNA plate for luminometric DNA-DNA hybridization to identify viridans streptococci in under 2 hours

Abstract Single-stranded chromosomal DNA was covalently bound to a microdilution plate and used for quantitative DNA-DNA hybridization. After 30 min, hybridized DNA was quantitatively detected by alkaline phosphatase and a chemiluminescent substrate. This method was successfully used for the rapid identification of viridans streptococci.     

Microencapsulation of Aerococcus viridans with catalase and its application for the synthesis of dihydroxyacetone phosphate

Dihydroxyacetone phosphate is essential for the synthesis of polyhydroxylated compounds used as components or precursors of active pharmaceutical substances, such as antibiotics or glycosidase inhibitors. Dihydroxyacetone phosphate was produced by enzymatic oxidation of L-alpha-glycerophosphate in the presence of glycerophosphate oxidase or Aerococcus viridans coimmobilized with a hydrogen peroxide-decomposing enzyme. The microencapsulation of A. viridans with catalase in sodium alginate showed a conversion of 98.5%; the conversion percentage remained constant in all five runs. Liquid chromatography of the product revealed that the product peak corresponded to that of the dihydroxyacetone phosphate internal standard. This indicated a high degree of product purity.     

Characterization of peptidoglycan stem lengths by solid-state 13C and 15N NMR

Lyophilized whole cells of Aerococcus viridans (Gaffkya homari) grown on a synthetic medium containing D-[2-13C, 15N]Ala, or containing both L-[1-13C]Lys and D-[15N]Ala, have been examined by double cross-polarization magic-angle spinning 13C and 15N nuclear magnetic resonance. Results from the double-labeled alanine experiment confirm the absence of metabolic scrambling of alanine by A. viridans. Results from the combined single-label experiment can be used to count directly the number of adjacent L-Lys and D-Ala units in peptide chains of cell-wall peptidoglycan. This count leads to the conclusion that there are no terminal D-Ala or D-Ala-D-Ala units in uncross-linked chains of the peptidoglycan of A. viridans.     

Quantitative polymerase chain reaction used for the rapid detection of Carnobacterium species from French soft cheeses

An identification method by PCR, specific to the Carnobacterium genus, was optimised by testing it on 28 bacterial strains. Primers from the literature were tested to differentiate Carnobacterium strains present among various bacterial species. The DNA of Carnobacterium species (C. alterfunditum, C. divergens, C. funditum, C. gallinarum, C. inhibens, C. maltaromaticum, C. mobile, C. viridans), specifically amplified by the Cb1-Cb2R primer couple at a hybridization temperature of 69 degrees C, gave only one band of 340 bp. The validation of this technique was carried out on a French soft cheese made with pasteurised milk inoculated with C. maltaromaticum LMA 28. Using classical PCR, detection was not possible for decimal dilutions of the cheese above 1 g L(-1). With Sybr Green I real time PCR, the specificity of the reaction was confirmed by the T(m) value. The standard curve constructed using the main threshold cycle and various concentrations of C. maltaromaticum LMA 28 (ranging from 10(0) to 10(8) cfu mL(-1)) showed good linearity and a sensitivity limit of > or = 10(4) cfu g(-1) of cheese. This technique was applied on commercially available cheeses made from raw cow's milk. The Sybr Green I real time PCR method constitutes an effective and easy-to-perform method to quantify Carnobacterium sp. in cheese.     

Bacteremia caused by viridans streptococci in 71 children.

A review of the hospital records of 71 patients from whose blood viridans streptococci were isolated showed that in 13 cases the patient''s illness was definitely related to the bacteremia: 4 patients had endocarditis, 3 had pneumonia, 2 had peritonitis and 1 each had meningitis, a scalp wound infection, sinusitis and otitis media. The bacteremia may have contributed to the two deaths among these 13 patients. In 45 cases the viridans streptococci may have contributed to the patient''s illness: 15 patients had an infection of the lower respiratory tract and 7 an infection of the upper respiratory tract, 8 were neonates with suspected septicemia, 3 had soft tissue infections, 3 had leukemia and sepsis, and 9 had miscellaneous infections; the bacteremia was unrelated to the two deaths in this group. In another 13 cases the viridans streptococci could not be related to the patient''s illness. The species most frequently isolated were Streptococcus mitis, S. sanguis II and S. MG-intermedius. The outcome of the bacteremia was generally good, even among the 11 patients not treated with antibiotics. When viridans streptococci are cultured from a single blood sample, further samples of blood and, if feasible, specimens from the associated focus of infection should be obtained for culture; further blood cultures are especially important in cases of suspected endocarditis.     

Non-linear slow-binding inhibition of Aerococcus viridans lactate oxidase by Cibacron Blue 3GA

Lactate oxidase (LOD) was purified from cells of Aerococcus viridans by phase partitioning in Triton X-114 (TX-114), ammonium sulphate fractionation and FPLC ion exchange chromatography. The purification achieved from a crude extract of A. viridans was 32-fold with a 60% recovery of activity. The isolated enzyme was a true FMN-containing LOD in tetrameric form with a subunit molecular weight of 48,000. The KM for L-lactate was 175 microM, a 6-fold less value than described in the literature. Among the inhibitors tested, Cibacron Blue 3GA showed the lowest Ki. At low concentrations, Cibacron Blue 3GA behaved as a dye-, pH- and time-dependent inhibitor. A Dixon plot of the steady-state rate showed the time-dependent inhibition to be non-linear, contrary to that described for other slow-binding inhibitors. A model to explain this phenomenon was proposed. The model implies the binding of Cibacron Blue 3GA to the isomerised form of the initial enzyme-inhibition complex (E'I).     

A rapid solid-phase fluorimetric assay for measuring bacterial adherence,using DNA-binding stains

In this report, we describe the validation of a rapid, single-step, microtiter plate method for quantifying bacterial adherence, based on fluorescent labeling of microorganisms with cell-permeable fluorescent DNA-binding probes. We have tested the binding to saliva-coated microtiter plates of bacteria, including Helicobacter pylori and viridans streptococci (S. mitis, S. gordonii, S. sanguis), known to interact with salivary components. Furthermore, we tested the short-term and longer-term temporal stability of a saliva-mediated adherence of these bacteria in a healthy population (N=30). The assay exhibited excellent reliability statistics, yielding within-assay variability coefficients ranging from 4.9% to 11%. A range of approximately 5 x 10(4)-1 x 10(7) cells could be detected. This method may be generally applicable to study surface binding of virtually any microbial species, while obviating the need of radioactive materials or specific antibodies for quantification, thus providing a procedure that is useful to both basic and clinical research.     

Antibacterial and antifungal lysozyme-type activity in Cameraria ohridella pupae

Lysozyme-type antibacterial and antifungal activity in pupae of Cameraria ohridella was studied. Activity against Micrococcus luteus and Bacillus megaterium was detected in pupae extract. Also antifungal activity from C. ohridella pupae extract directed against Saccharomyces cerevisiae strain W 303 was shown. During immunoblotting two bands in pupae extract, with molecular mass of about 15 and 28 kDa were recognized by antibodies directed against HEWL. After acid electrophoresis followed by bioautography of the extract, two lytic zones showing lysozyme-type activity against M. luteus were observed. Two bacteria: Gram-positive Aerococcus viridans and Gram-negative Aeromonas salmonicida ssp. masoucida were isolated from pupae of C. ohridella. Their activity against M. luteus, B. megaterium, and S. cerevisiae W303 was detected. After immunoblotting with antibodies against HEWL, also two proteins from bacterial suspensions of A. viridans and A. salmonicida were detected, about 15 and 28 kDa.