Characterization of Aerococcus viridans isolated from milk samples from cows with mastitis and manure samples

Thirty-eight Aerococcus viridans isolates were obtained from milk from 478 cows with clinical mastitis in a farm during the periods between November 2011 and February 2012, and between December 2012 and March 2013. Additional isolates were obtained from processed manure (a mixture of composted manure, straw and hydrated lime) and bedding materials. The processed manure was later used to cover the floor of the stalls in barns as bedding materials. The temperatures recorded in the composted and processed manure were not as high as those generally observed during satisfactory composting. To reveal the association of A. viridans in manure-related products with intramammary infection in cows, isolates were characterized by their DNA fragment patterns as determined by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. Isolates obtained from milk, processed manure and bedding materials had identical DNA fragment patterns. Antimicrobial susceptibilities were determined for 29 isolates from milk, processed manure and bedding materials. Of these, 26 (89.7%) were resistant to clindamycin, whereas virtually all the isolates were susceptible to 12 other antimicrobials including cefalosporins that have been used to treat bovine mastitis in Japan. In vitro, three A. viridans isolates from milk and an isolate from processed manure survived for 3 hr in Good’s buffer (pH 9) at high temperature (50°C). The results suggest that the processed manure and bedding materials in this farm were possible sources of A. viridans that caused infection in the cows with mastitis.     

Isolation,Identification, 16S rRNA Gene Sequencing and Genetic Evolution Analysis of Aeromonas aeruginosa from Duck

【Objective】 The experiment was aimed to study the pathogenic bacteria of acute death of ducklings in a duck farm in Henan province and its phylogenetic status.【Method】 The liver and spleen of dead ducks in the diseased duck farm were collected.The bacterial morphology, Gram-staining, biochemical characteristics, sequence analysis of 16S rRNA gene, drug sensitivity test and pathogenicity test were carried out.【Results】 The isolated bacteria grew on blood agar medium with smooth, convex, milky white and α-hemolytic Gram-positive cocci.Biochemical test results showed that the isolated bacteria were positive for maltose, sucrose, raffinose, nitrate reduction reaction, MP-VP test, urea, lysine decarboxylase, ornithine decarboxylase and aescin, and negative for xylose, lactose, glucose, sorbitol, hydrogen sulfide, citrate, peptone, mannitol, phenylalanine and methyl red.The results of 16S rRNA gene sequencing and BLAST comparison results showed that the 16S rRNA gene sequence of the isolated strain was 94.8%-99.9% similar to that of Aerococcus viridans published on NCBI, and the similarity with strains Mnlv1, Mnlv2 and W66 was the highest, up to 99.9%.The similarity with strain GXBl-1 was the lowest, which was 94.8%.Phylogenetic tree analysis showed that the isolated bacteria belonged to the same genus and group on the same branch as strains of Aerococcus viridans FL09, 15MS and Mnlv2 etc., and had the closest genetic relationship with FL09.The results of drug sensitivity showed that the isolated strain was sensitive to fosfomycin, ampicillin and amoxicillin, moderately sensitive to penicillin, neomycin and amikacin, it was resistant to rifampicin, neomycin, bacitracin, erythromycin and enrofloxacin.The results of pathogenicity test showed that the strain was lethal and increased with the increase of inoculation concentration.【Conclusion】 This study determined that the pathogen causing the acute death of ducklings in a duck farm in Henan province was Aerococcus viridans, which provided a reference basis for clinical diagnosis and treatment of the diseases caused by this bacteria.     

Aerococcus viridans expression of Cpn60 is associated with virulence during infection of the American lobster, Homarus americanus Milne Edwards

The Gram-positive bacterium Aerococcus viridans var. homari is a well-documented causative agent of the lethal systemic disease gaffkemia in both the American lobster, Homarus americanus, and the European lobster, Homarus gammarus. Previous phenotypic characterization has been unsuccessful at differentiating avirulent from virulent strains without performing lethal animal infection trials. Recent genetic characterization of A. viridans strains through 16S rRNA sequencing and random amplification of polymorphic DNA fingerprinting has revealed the presence of two subtypes. However, subtype 1 contains both virulent and avirulent strains which are genetically identical. The purpose of this study was to determine the proteomic mediators of virulence in A. viridans. Quantitative proteomic mapping of these two strains has revealed 29 differentially expressed protein spots, seven of which are only expressed in the virulent strain and could act as virulence factors. One protein, chaperonin 60 (Cpn60), is uniquely expressed in the virulent strain and has been shown to act as a virulence factor in many other bacteria. The proteomic mapping strategy employed in this study is the first to show phenotypic differences between virulent and avirulent strains. Cpn60 expression represents a potentially useful tool for identifying the virulent strains of A. viridans in epidemiological studies.     

3株猪源绿色气球菌的分离鉴定

为确定云南省不同地区3个猪场仔猪发生四肢运动障碍的病原因素,无菌采集病死仔猪内部组织样品,采用血琼脂分离培养病原菌,随后对分离菌株做形态观察、生化试验、药敏试验及16S rRNA基因测序分析。结果：从3个猪场病料中均分离到菌落及个体形态一致的细菌,其16S rRNA基因序列与NCBI中7株绿色气球菌参考菌株的同源性均在99%以上,据此判定为绿色气球菌(Aerococcus viridans);3个分离菌株均对克拉霉素、头孢曲松和青霉素敏感,对复方新诺明、庆大霉素和卡那霉素耐药。鉴于病死猪有检出其他病原体感染的情况和未见该菌单独有强致病力的报道,研究结果提示绿色气球菌是危害猪健康养殖的重要机会致病菌。     

竹鼠源绿色气球菌的分离鉴定及耐药性分析

为了查明造成贵州某竹鼠养殖场竹鼠死亡病因,本研究从送检竹鼠体内分离到1株细菌（GZ-S1）,通过形态学观察、生化试验、16S rRNA基因序列分析、药敏试验及耐药基因检测、细菌致病性试验等方法对该分离菌进行鉴定。结果显示,该分离菌在鲜血琼脂平板上长出凸起、表面光滑、白色、呈α-溶血的革兰氏阳性球菌;生化试验显示该分离菌对蔗糖、果糖、葡萄糖、硝酸盐还原试验、靛基质试验、枸橼酸盐试验等均为阳性,半乳糖、甘露糖、硫化氢试验、VP试验、MR试验、赖氨酸脱羧酶接触试验等均为阴性;经16S rRNA基因测序及BLAST比对、系统进化树分析发现,分离菌株GZ-S1与气球菌菌株201707CJKOP-Y31（MG593595.1）、绿色气球菌菌株Mnlv2（GQ246745.1）等12株参考菌株在同一分支上。细菌药敏试验结果显示,分离菌株对苯唑西林、羧苄西林、头孢曲松3种药物敏感;对阿莫西林、氧氟沙星、复方新诺明、磺胺异噁唑、多黏菌素B、氟苯尼考、头孢拉定7种药物中敏;对恩诺沙星、卡那霉素、妥布霉素、新霉素、多西环素、四环素6种药物耐药。耐药基因检测结果显示,该分离菌株携带5种耐药基因：tetA（1 031 bp）、tetB（513 bp）、tetD（326 bp）、aadd（146 bp）、aac（6'）Ⅰb（655 bp）,与药敏表现型相符。人工感染试验结果显示,该分离菌有致病力。本研究对某竹鼠养殖场竹鼠发病的原因作出了准确诊断,为该养殖场细菌性竹鼠病的预防和合理用药提供参考。     

随机扩增多态性DNA技术对绿色气球菌的相关性分析

为考察恩诺沙星与乙酰甲喹的联合抗菌活性,采用肉汤稀释棋盘法测定了恩诺沙星与乙酰甲喹单用及联用对大肠杆菌、沙门氏菌临床菌株的体外抗菌活性。结果显示:恩诺沙星与乙酰甲喹单独使用对大肠杆菌的最小抑菌浓度为8μg/m L和32μg/m L,对沙门氏菌的最小抑菌浓度为16μg/m L和256μg/m L。两药联用对大肠杆菌和沙门氏菌的FIC指数为0.5和0.75,分别表现为协同作用和相加作用。试验表明,恩诺沙星与乙酰甲喹联用可以增强对大肠杆菌和沙门氏菌感染的治疗效果。     

猪源绿色气球菌的分离鉴定及16S rRNA基因序列测定与遗传进化分析

为研究引起广东部分地区猪场仔猪发生急性死亡的致病菌及其系统发育地位,本研究从广东英德、惠州龙门、翁源、汕头等地区猪场急性死亡病仔猪中分离到6株呈α溶血的细菌。通过细菌形态学观察、生化特性鉴定、药敏试验、致病性试验以及16S rRNA基因的序列分析,确定为猪源绿色气球菌,分别命名为HD、WY、ZQ、LM、YD和ST。动物感染试验表明：6株分离菌株均具有不同程度的致病性;药敏结果显示：6株分离株均对头孢曲松、头孢唑啉、恩诺沙星、阿莫西林高度敏感,对新生霉素、利福平、阿米卡星、庆大霉素、多粘菌素B中度敏感,对其他抗生素均耐药;同源性及遗传进化分析结果显示：6株分离株与12株国内外参考菌株核苷酸同源性在93%以上,其中与澳大利亚分离自犬的15MS株同源性最高,为98.1%,与广西分离自猪的GXBL-1株同源性最低,为83.6%;6株分离株与广东分离株和澳大利亚分离株亲缘关系最近,暗示进化不存在明显的地域相关性。本研究确定了广东部分地区引起规模化猪场仔猪发生急性死亡的病原菌,为该菌引起的疾病的防治提供了依据。