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.     

Purification and properties of Aerococcus viridans lactate oxidase

Lactate oxidase was purified from cells of Aerococcus viridans by a procedure which utilized ammonium sulfate fractionation, DEAE Sepharose CL-6B chromatography, and Sephadex G-100 chromatography. The final preparation was homogeneous by SDS-polyacrylamide gel electrophoresis. The enzyme appears to be a tetramer with a subunit molecular weight of 44,000 and utilizes FMN as a cofactor. The enzyme was highly specific for L-lactate. D-lactate, glycolate, and D,L-2-hydroxybutyrate were not oxidized by the enzyme but were competitive inhibitors. The enzyme could be irreversibly inactivated by incubation with bromopyruvate. This inactivation appears to involve a covalent modification near the active site of the enzyme; however, the flavin cofactor is not the site of this modification.     

The lactate oxidase activity of Aerococcus viridans cultures

The oxidase activity of Aerococcus viridans is a result of the functioning of aerobic NAD-independent lactate dehydrogenase. Two fractions of flavin-containing protein which oxidize D- and L-isomers of lactic acid are revealed during the enzymic complex electrophoresis in PAAG. The enzymic complex and eluates of both fractions possess the antagonistic activity relative to 27 test-cultures of bacteria.     

眼部浅绿色气球菌感染4例报道

目的 提高对眼部气球菌感染的认识.方法 报告4例眼部气球菌感染病例并复习文献,对其发病因素、临床症状、诊断、及治疗和预后进行分析.结果 4例眼部感染气球菌患者为中年或老年人,基础疾病为青光眼、糖尿病(2例)及甲状腺功能亢进症,均经病原菌的分离鉴定后确诊为浅绿色气球菌感染,且对左氧氟沙星、万古霉素等常用药敏感,4例患者中除1例青光眼患者用药效果不理想外,其余均经1～2周的治疗后痊愈.结论 对眼部感染患者,应加强浅绿色气球菌的分离与鉴定.     

The adhesive and colonizing properties of Aerococcus viridans]

On the basis of experiments carried out in the course of this study the conclusion can be made that one of the mechanisms of the preventive effect of aerococci, the representatives of the indigenous microflora of the gastrointestinal tract, lies in their capacity for adhesion to and colonization of the mucous membrane, this enhancing the resistance of the enteric tract to infections. These properties of anterococci substantiate good prospects of using M-bacterin (live lyophilized aerococcal culture) for the control of enteric infections and dysbacteriosis.     

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.     

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.     

Molecular and Phenotypic Characterization of Aerococcus viridans Associated with Subclinical Bovine Mastitis

Aerococcus viridans is a wide spread bacterium in the environment and clinically this organism is associated with different diseases in animals and humans. However, the geno- and phenotypic characterization of A. viridans associated with bovine mastitis has not yet been reported. The objectives of this study were to investigate the genetic and phenotypic diversity of A. viridans isolates using three different molecular methods including 16S rRNA gene sequencing, pulsed-field gel electrophoresis and random amplified polymorphic DNA (RAPD) along with biochemical tests, including antimicrobial susceptibility test. In total, 60 A. viridans strains were cultured from dairy herds presenting with subclinical mastitis. The results of biochemical tests revealed that most of the isolates (75.0%) were accurately identified by API Rapid 20 Strep system and the majority of A. viridans strains (96.7%) were found to be catalase negative, while two (3.3%) isolates were weakly positive. All isolates were resistant to trimethoprim-sulfamethoxazole, followed by streptomycin (96.7%), tetracycline (65.0%) and clindamycin (56.7%) by minimum inhibition concentration-determining broth microdilution technique. As compared to the sequence of 16S rRNA gene, both PFGE and RAPD showed their capacities to discriminate the intra-species diversity of A. viridans. Furthermore, most of the isolates obtained from the same herd or region belonged to the same major RAPD group, which indicated that RAPD is an appropriate assay for tracking the origins of isolates and epidemiological studies of A. viridans. This is a novel approach to use three molecular techniques and to compare their efficiency regarding the genetic diversity of A. viridans. The data suggest that A. viridans associated with subclinical mastitis has a considerable phenotypic and genotypic diversity.     

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.     

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).     

Properties and stability of glycerophosphate oxidase isolated from a mutant strain of Aerococcus viridans

The properties of microbial L-alpha-glycerophosphate oxidase (GPO) isolated from a mutant strain of Aerococcus viridans DBM 1509 were estimated. The stability at different temperatures and pH were detected. At 4 degrees C the enzyme lost activity during 15 d, at 20 degrees C and 30 degrees C GPO activity decreased during 30 and 25 h, respectively. The highest stability was measured at - 20 degrees C and pH 9. At 4 degrees C the stability was enhanced by the addition of 0.1 M EDTA or by lyophilization in the presence of dextrin. These conditions allow the prolongation of the low stability of microbial GPO which limited its use, and give the opportunity to increase the stability of other enzymes     

A Study on the Kinetic Mechanism of Apoenzyme Reconstitution from Aerococcus viridans Lactate Oxidase

The preparation of a reconstitutable apoprotein is widely recognized as an important tool for studying the interactions between protein and coenzyme and also for characterizing the coenzyme-binding site of the protein. Here is described the kinetic analysis of the reconstitution of Aerococcus viridans lactate oxidase apoenzyme with FMN and FAD in the presence of substrate. The reconstitution was followed by measuring the increase in catalytic capacity with time. Lactate oxidase activity was easily removed by obtaining its apoenzyme in an acidic saturated ammonium sulphate solution. When the apoenzyme was reconstituted by the addition of FMN or FAD, a marked lag period was observed, after which the system reached a steady state (linear rate). To explain the binding mechanism of the cofactors to the apoenzyme, a kinetic model is proposed, in which the constants, k₃ and k_-3, representing the interaction of apoenzyme with cofactor are considered slow and responsible for the lag in the expression of activity. The affinity of apoenzyme was 51-fold higher for FMN than FAD.     

Rapid identification of Aerococcus viridans using the polymerase chain reaction and an oligonucleotide probe

A polymerase chain reaction/oligonucleotide probe method was developed for the specific identification of the Gram-positive bacterium Aerococcus viridans. Primers for the enzymatic amplification reaction were designed from specific sequences within the 16S rRNA. The method was also highly sensitive and 10 cfu of A. viridans could be detected in 5 h although the reliability of detection was poor in mixed cultures with Escherichia coli.     

A study on the kinetic mechanism of apoenzyme reconstitution from Aerococcus viridans lactate oxidase

The preparation of a reconstitutable apoprotein is widely recognized as an important tool for studying the interactions between protein and coenzyme and also for characterizing the coenzyme-binding site of the protein. Here is described the kinetic analysis of the reconstitution of Aerococcus viridans lactate oxidase apoenzyme with FMN and FAD in the presence of substrate. The reconstitution was followed by measuring the increase in catalytic capacity with time. Lactate oxidase activity was easily removed by obtaining its apoenzyme in an acidic saturated ammonium sulphate solution. When the apoenzyme was reconstituted by the addition of FMN or FAD, a marked lag period was observed, after which the system reached a steady state (linear rate). To explain the binding mechanism of the cofactors to the apoenzyme, a kinetic model is proposed, in which the constants, k3 and k-3, representing the interaction of apoenzyme with cofactor are considered slow and responsible for the lag in the expression of activity. The affinity of apoenzyme was 51-fold higher for FMN than FAD.     

猪源绿色气球菌的分离鉴定与药敏特性研究

目的鉴定研究2006年和2008年本实验室分别从广东清远和广西北流两个猪场发病小猪的膝关节积液中分离到2株呈四联状或成对排列,在山羊血琼脂平板上能形成明显的α-溶血圈的G+球菌。方法采用小白鼠致病性试验、细菌常规生理生化鉴定、药敏试验、耐药基因检测及16S rRNA序列分析等方法对上述分离的2株细菌进行鉴定及药敏特性研究。结果确定这2株G+球菌为正常情况下非致病性绿色气球菌(登录号分别为:GQ161096;GQ161097)。系统发育分析表明,两分离株与绿色气球菌15MS(登录号:EU075039.1)同源性分别高达99.4%和98.7%。药敏试验及耐药基因检测发现,这2株绿色气球菌除了对新霉素、氟哌酸、恩诺沙星、菌必治和环丙沙星等5种药物呈高敏外,对先锋V等13种抗菌素均不敏感。耐药基因检测结果表明,GXBL-1可检出6个耐药基因,GDQY-1则可检出多达10个耐药基因。结论从这2株分离株耐药谱检测结果可以看出,正常菌株同样携带多重耐药基因,存在着通过质粒转座子和整合子将耐药基因转移给敏感菌,导致耐药性传播的可能,应引起足够重视。     

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.