石鲽病原琼氏不动杆菌形态型Ⅰ的鉴定*

从由杀鲑气单胞菌(Aerom onas salmonicida)所引起的石鲽(Stone flounder,Kareiusbicoloratus L.)细菌性败血感染症病(死)鱼肝、脾、肾及肠内容物中,同时检出了作为继发感染的病原菌。经对6株纯培养菌在形态特征、理化特性等方面较系统的表观分类学指征鉴定及代表菌株DNA中G+C mol%的测定,表明为琼氏不动杆菌的一个新形态型并定名为琼氏不动杆菌形态型Ⅰ(Acinetobacter juniimorphovarⅠ);     

石鲽细菌性败血感染症及其病原细菌研究

2001—2002年,分别对两起养殖的石鲽(Stone flounder,Kareius bicoloratusL.)病害进行了检验,均表现为败血症感染特征。经以20尾病(死)石鲽(每起病例各10尾)做病变组织中细菌检查、细菌分离与鉴定、人工感染试验等的病原学检验,表明两起被检病例均为同种的杀鲑气单胞菌(Aeromonas salmonicida)感染;另在其中1起病例所检10尾石鲽的2尾中,同时检出了呈继发感染的不动杆菌(Acinetobacter)。通过对所分离后做纯培养的60株杀鲑气单胞菌6、株不动杆菌分别进行较系统的表观分类学指征(Phenotypic information)及代表菌株16S rRNA基因序列测定与系统发育学分析,表明其中的杀鲑气单胞菌为一个新亚种,不动杆菌为琼氏不动杆菌(Acinetobacter junii)的一个新形态型;又择代表菌株送中国典型培养物保藏中心(CCTCC)进行了表观性状、DNA中C Cmol%的测定等的复核鉴定,并将其中的杀鲑气单胞菌新亚种定名为杀鲑气单胞菌杀鲽亚种(Aeromonas salmonicidasubsp.flounderacidasubsp.no...     

鲍曼不动杆菌耐药持留菌的特征及Ⅱ型毒素-抗毒素系统的多样性

摘要:【目的】研究鲍曼不动杆菌持留菌的特征,分析鲍曼不动杆菌Ⅱ型毒素-抗毒素(TA)系统的多样性及分布,探讨TA系统与持留菌形成的潜在关联。【方法】采用分属于6 大类中的6种不同抗生素分别筛选的方法分离并定量鲍曼不动杆菌的持留菌;应用PSI-BLAST及TBLASTN程序分析721株鲍曼不动杆菌中的Ⅱ型TA系统;通过聚合酶链式反应(PCR)方法检测44株临床多重耐药(MDR)的鲍曼不动杆菌中5个Ⅱ型TA系统的分布。【结果】我们发现不同抗生素处理得到的鲍曼不动杆菌持留菌的数量并不相同;对多数抗生素而言,浓度越高,得到的持留菌数量越少;处于生长平台期的菌株中相比对数期含有更多的持留菌;多粘菌素B及妥布霉素均具有杀灭部分持留菌的能力;鲍曼不动杆菌所有菌株中均含有Ⅱ型TA系统,HTH/GNAT类型在菌株中分布最为广泛;所有700余株鲍曼不动杆菌基因组中共含有15个潜在的Ⅱ型TA系统;三类普遍存在于已知基因组中的明确功能的Ⅱ型TA系统在临床多重耐药菌株中同样分布广泛,且HigB/HigA的表达量在标准株的持留菌中显著增高。【结论】鲍曼不动杆菌的持留菌水平与菌株的生长状态、抗生素的种类及浓度密切相关。Ⅱ型TA系统在鲍曼不动杆菌已知基因组和临床多重耐药菌株中普遍存在,其中,HTH/GNAT、GP49/Cro(HigB/HigA)及DUF497/COG3514三种类型的Ⅱ型TA系统可能在鲍曼不动杆菌持留菌的形成中扮演重要角色。     

几种尖孢镰刀菌专化型中SIX1、SIX4、SIX6、SIX8同源基因分析

尖孢镰刀菌在与寄主的相互作用中分泌几个特定的富含半胱氨酸的小分子量蛋白进入木质部中启动致病力,被称为SIX(secreted in xylem)蛋白,为明确其在不同寄主中的作用,本研究比较分析了几种尖孢镰刀菌专化型中SIX1、SIX4、SIX6、SIX8同源基因序列。根据已完成的尖孢镰刀菌古巴专化型1号(Foc1)与4号生理小种(Foc4)全基因组测序序列信息及相关SIX基因序列设计引物,应用PCR方法扩增分析56株尖孢镰刀菌古巴专化型与18株其它专化型及非致病型尖孢镰刀菌菌与其它种或属共21株菌株中的SIX1、SIX4、SIX6、SIX8基因。结果表明:设计的SIX1、SIX4、SIX6、SIX8基因引物均不能从非致病性尖孢镰刀菌与其它镰刀属种或其它属的菌株DNA中扩增出目的条带;SIX1基因的2个引物均能从供试的Foc菌株DNA中扩增出目的条带,同时可从部分其它专化型菌株DNA中扩增出目的条带;SIX4基因引物仅能从供试的尖孢镰刀菌番茄专化型与部分甘蓝专化型菌株DNA中扩增出目的条带;SIX6引物仅能从供试Foc1、Foc2、Foc4菌株DNA中扩增出目的条带;SIX8基因引物能从所有供试的致病尖孢镰刀菌中DNA扩增出目的条带。研究发现的SIX6基因序列提供了快速鉴定尖孢镰刀菌古巴专化型的检测方法,同时为深入研究尖孢镰刀菌各个专化型中SIX基因的功能奠定基础。     

脂肪酸组分分析在不动杆菌鉴定中的应用

以不动杆菌属(Acinetobacter)中的A. baumannii、A. haemolyticus、A. johnsonii、A. junii、A. lwoffii模式菌株为参比菌株,对中国农业微生物菌种保藏管理中心(ACCC)所保藏的6株不动杆菌进行脂肪酸组分分析及16S rRNA基因系统进化分析.结果表明,利用脂肪酸分析可将6株菌完全准确地鉴定到属的水平上,这一点与16S rRNA基因分析结果一致;在种的水平上,16S rRNA基因进化分析与脂肪酸组分分析的结果可互为补充,相互印证.同时,通过对不动杆菌部分模式菌株及供试菌株的脂肪酸组分分析,报道了不动杆菌的特征性脂肪酸为C18:1 w9c、C16:00和C16:1w7c/C16:1w6c.     

牙鲆病原秦皇岛弧菌主要生物学性状研究

从秦皇岛某海水养殖场牙鲆(ParalichthysolivaceusL.)细菌性败血感染症的病例中,分离到了相应的病原细菌。经对12株纯培养菌进行形态特征、培养特性、理化特性等方面较系统的表观分类学指征及代表菌株DNA中G+Cmol%的测定,表明为弧菌属(VibrioPacini1854)细菌的一个新种,并定名为秦皇岛弧菌(Vibrioqinhuangdaorasp.nov.)。同时对该菌的血清型进行了检定,表明12株菌具有同种的K抗原和同种的O抗原(血清同源);另外,经以37种抗菌类药物做敏感性测定,结果显示对供试的头孢唑啉等32种药物敏感、对青霉素G等5种耐药,在不同菌株间无明显的敏感与耐药差异。       

鲍曼不动杆菌IRS-PCR基因分型研究

目的 用低频限制性位点聚合酶链反应(IRS-PCR)对鲍曼不动杆菌进行基因分型,分析基因型与鲍曼不动杆菌耐药谱的关系,并初步探讨其在分子流行病学中的作用.方法 随机收集2008年8月至2009年8月临床分离的73株鲍曼不动杆菌,采用K-B法进行药物敏感试验确定鲍曼不动杆菌耐药谱;同时利用IRS-PCR对此73株鲍曼不动杆菌进行基因分型;并分析IRS-PCR分型与鲍曼不动耐药谱的关系;结合IRS-PCR分型结果与73株鲍曼不动杆菌感染病例的临床资料,分析在此时间段鲍曼不动杆菌在我院流行感染的情况.结果 药物敏感试验将73株鲍曼不动杆菌菌株分为A1(19株全耐药型)和A2 ～ A31(54株耐药谱型)31个药敏谱.IRS-PCR法将其分为A～W共23个基因型,其中A、C、B、D和E型为5种优势菌株,分别为14、11、10、8和6株.对比研究发现A1型菌株(15/19)主要集中在基因型A、C、D内,而基因型B包含A15型耐药菌株9株(69.2％),基因型E包含A3型耐药菌株3株(42.9％).A基因型在院内特别是ICU中心引起2次爆发流行,而C和D型主要在呼吸内科引起感染.结论 IRS-PCR基因分型与药敏分型有较高的一致性,且IRS-PCR基因分型在早期发现和预防感染暴发流行方面优于药敏分型.     

锦鲤中吉氏库特菌的分离与鉴定

对从患病锦鲤(Cyprinus carpio L.)肝组织中分离的1株纯培养菌(HC050630C-1)进行了形态特征、主要理化特性、对健康鲤鱼的致病作用、药物敏感性等方面的检验;同时测定了该株菌的16S rRNA基因序列,构建了系统发育树。结果表明,被检菌为吉氏库特菌(Kurthia gibsonll),对健康鲤鱼未显示明显的致病作用;所测菌株的16S rRNA基因序列长度1459bp,在GenBank中登录号为EF611423;该菌株16SrRNA基因序列与GenBank数据库中吉氏库特菌的16S rRNA基因序列同源性在99%;药敏试验结果显示,对供试的氨苄青霉素等27种药物呈现高度敏感或敏感,对头孢吡肟等6种呈低度敏感,对苯唑青霉素等4种耐药。     

牙鲆迟钝爱德华氏菌感染症及其病原的研究

对 7起牙鲆迟钝爱德华氏菌感染病例进行了发病情况、临床特征、病理变化等方面的检验 ,经对细菌的分离与鉴定表明所检病例均为迟钝爱德华氏菌的单独感染 ,系统归纳了该感染症的主要特点。同时 ,对所分离后做纯培养的 130株迟钝爱德华氏菌进行了主要生物学性状、血清型的测定 ,表明除在生化试验的吲哚项目中表明有株间差异 (阴性的 2 0株、阳性的 110株 )外 ,130株对其他所测内容的结果一致 ,130株均为同种血清型。从每起病例分离并鉴定的各 1个代表菌株做对健康牙鲆的人工感染试验 ,表明了相应的原发病原学意义及较强的致病作用。药敏试验结果表明 ,对供试 37种抗菌药物中的头孢唑啉等 19种药物敏感、对青霉素G等 5种药物耐药、对氨苄青霉素等 13种药物表现了株间差异。经以荧光抗体技术对纯培养物、人工感染病死鱼肝脏中细菌的检验 ,初步表明了荧光抗体技术在对迟钝爱德华氏菌检验中作为辅助检验手段的可行性。     

100例鲍曼不动杆菌的耐药性与整合子表达及耐药基因分析

目的 了解鲍曼不动杆菌的耐药性和整合子表达及耐药基因携带情况.方法 收集100株鲍曼不动杆菌,以VITEK-64系统鉴定细菌,并进行14种抗生素药敏试验,通过PCR法检测Ⅰ、Ⅱ、Ⅲ类整合酶基因(intI1、2、3)及Ⅰ类整合子可变区基因盒,并对基因盒测序.结果 除阿米卡星和头孢哌酮/舒巴坦,鲍曼不动杆菌对其他12种抗菌药物耐药率均大于60.0％,多重耐药率为88.0％.鲍曼不动杆菌整合酶基因阳性率为64.0％,均为intI1,整合子阳性菌株对多数药物的耐药率显著高于整合子阴性者(P＜0.05).intI1阳性菌株中,84.4％ (54/64)扩增出整合子可变区,检出3种耐药基因盒组合形式:aac(6’)-Ib-cr-arr-3-dfrA27 14株、aacA4-catB8-aadA1 24株、aacC1-orfA-orfB-aadA1 16株.结论 临床分离的鲍曼不动杆菌多重耐药与Ⅰ类整合子表达有关.Ⅰ类整合子主要携带早期使用的氨基糖苷类抗菌药、甲氧苄啶和氯霉素耐药基因.     

Coaggregation among nonflocculating bacteria isolated from activated sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.     

Adhesion of Acinetobacter spp. to para-xylene

Hydrophobicity of 200 of Acinetobacter spp. strains was defined by the BATH (Bacterial Adhesion to Hydrocarbon Test) method of Rosenberg et al. 48.0% strains of Acinetobacter spp. adhered to para-xylene. 40.0% of strains showed waek hydrophobicity, 7.5% moderate and one strain strongly adhered to para-xylene. A. baumannii strains adhered in 52.2%, A. junii in 42.1% and 20.0% in A. haemolyticus. Significantly we find weak adhesion to para-xylene than mild and strong. Mostly adhesion was discovered in A. baumannii and A. junii than in A. haemolyticus. Results show that Acinetobacter spp. has adhesive properties to para-xylene.     

Intergeneric coaggregations among Oligotropha carboxidovorans and Acinetobacter species present in activated sludge

The coaggregation traits of two pairs of sewage sludge bacteria were tested and characterized. Oligotropha carboxidovorans S23 coaggregated with two strains of the genus Acinetobacter viz. Acinetobacter junii S33 (56%) and Acinetobacter johnsonii S35 (99%). Coaggregates of O. carboxidovorans S23 and A. junii S33 were small (20-40 microm), weak and susceptible to EDTA and a commercial protease (Actinase E). Actinase/periodate pretreatment of the partners prior to coaggregation revealed that interaction in this case was mediated by protein surface components. Coaggregates of O. carboxidovorans S23 and A. johnsonii S35 were large (above 100 microm), strong and not deflocculated by EDTA or Actinase E. Only periodate pretreatment of A. johnsonii S35 prevented this coaggregation indicating a role for a carbohydrate-containing moiety without the involvement of protein components. The potential mechanisms and strength of bacterial coaggregations seem to be pair dependent.     

The pathogenicity factors of bacteria in the genus Acinetobacter]

Eighty Acinetobacter strains, isolated in Togliatti from patients with purulent inflammatory diseases, were studied to determine their pathogenicity factors. Out of these 80 strains, 32.5% were found to have enterotoxigenic activity and 46.2%, adhesive activity. They were related to adhesins of the human type and to adhesins of the sheep, rabbit, swine and guinea pig types. But the most important phenomenon established in this study was the combination of different pathogenicity factors detected in Acinetobacter bacteria. Analysis of the combination of pathogenicity factors revealed that 7.5% of Acinetobacter strains had adhesive and enterotoxigenic activity, 15.3% of these strains combined adhesive and hemolytic activity and 1.2% of them were found to be enterotoxigenic and hemolytic. Only 5.0% of Acinetobacter strains were found to carry all there pathogenicity factors simultaneously.     

Adhesins of Acinetobacter strains

One of the important factors contributing to the pathogenicity of bacteria is the presence of adhesins on cell surface, which facilitate colonisation in the macroorganism. The presence and type of adhesins occurring in four species of the genus Acinetobacter: A. baumannii (184), A. junii (59), A. lwoffii (65) and A. haemolyticus (22) was determined by haemagglutination test with a 3% suspension of fresh, tannic acid-treated of guinea pig, cow and human group O and AB erythrocytes, with or without the addition of one of sugar inhibitors (D-mannose, alpha-methylmannopyranoside, D-galactose-N-acetyl-D-glucosamine, L-fucose and D-ribose). In strains from all species, adhesines of the mannose-resistant (MR) type dominated. The mannose-sensitive (MS) type was present solely on the surface of one A. lwoffii strains. A. baumannii (36), A. junii (8), A. lwoffii (11) and A. haemolyticus (4) exhibited mannose-resistant hemagglutination in relation to fresh erythrocytes and that reaction was restrained by D-galactose, D-galactose and L-fucose (no other inhibitor used restrained it). The results achieved prove that cell adhesines other than those of MR type must be present on the cell surface. Additional adhesines occurred mainly in strains isolated from the respiratory and urinary tract infection simples, but were not found in isolates from blood cultures.     

ARDRA联合RAPD对不动杆菌基因型鉴定的研究

收集多重耐药的不动杆菌10株,以标准参照株作对照,采用扩增核糖体DNA限制性酶切(ARDRA)DNA指纹技术联合随机扩增多态性DNA(RAPD)技术对其基因亚型进行分析;以非加权组间平均法(UPG-MA)进行聚类分析。该法可以有效地鉴定不动杆菌基因亚型;并从10株不动杆菌中鉴定出1株琼氏不动杆菌及9株鲍曼不动杆菌。ARDRA联合RAPD基因指纹分型技术有良好的互补性,可准确鉴定不动杆菌基因型。     

适于罗布麻生物脱胶的果胶酶产生菌分离筛选与表征

在青海柴达木盆地沤制的罗布麻表皮中分离得到两株适于罗布麻脱胶的高产果胶酶菌株,经形态、生理生化指标鉴证和16SrDNA菌种鉴定,确定一株为新的果胶酶产生菌琼氏不动杆菌（Acinetobacter junii）,一株为枯草芽孢杆菌（Bacillus subtilis）。水解圈实验得出前者H／C值（H／C为水解圈直径H与菌落直径C之比）为5,后者为3;经酶活力测定,在37℃下,琼氏不动杆菌（Acinetobacter junii）在11h达到产酶高峰（酶活力为103．2IU／mL）,枯草芽孢杆菌（Bacillus subtilis）培养至9h达到产酶高峰（酶活力为91．6IU／mL）,但前者最高酶活力比后者高12．5％;经脱胶实验得出两菌残胶率分别为18．47％和17．31％,对罗布麻生物脱胶有较好的适用性。     

Comparison of the virulence potential of Acinetobacter strains from clinical and environmental sources

Several Acinetobacter strains have utility for biotechnology applications, yet some are opportunistic pathogens. We compared strains of seven Acinetobacter species (baumannii, Ab; calcoaceticus, Ac; guillouiae, Ag; haemolyticus, Ah; lwoffii, Al; junii, Aj; and venetianus, Av-RAG-1) for their potential virulence attributes, including proliferation in mammalian cell conditions, haemolytic/cytolytic activity, ability to elicit inflammatory signals, and antibiotic susceptibility. Only Ah grew at 10(2) and 10(4) bacteria/well in mammalian cell culture medium at 37°C. However, co-culture with colonic epithelial cells (HT29) improved growth of all bacterial strains, except Av-RAG-1. Cytotoxicity of Ab and Ah toward HT29 was at least double that of other test bacteria. These effects included bacterial adherence, loss of metabolism, substrate detachment, and cytolysis. Only Ab and Ah exhibited resistance to killing by macrophage-like J774A.1 cells. Haemolytic activity of Ah and Av-RAG-1 was strong, but undetectable for other strains. When killed with an antibiotic, Ab, Ah, Aj and Av-RAG-1 induced 3 to 9-fold elevated HT29 interleukin (IL)-8 levels. However, none of the strains altered levels of J774A.1 pro-inflammatory cytokines (IL-1β, IL-6 and tumor necrosis factor-α). Antibiotic susceptibility profiling showed that Ab, Ag and Aj were viable at low concentrations of some antibiotics. All strains were positive for virulence factor genes ompA and epsA, and negative for mutations in gyrA and parC genes that convey fluoroquinolone resistance. The data demonstrate that Av-RAG-1, Ag and Al lack some potentially harmful characteristics compared to other Acinetobacter strains tested, but the biotechnology candidate Av-RAG-1 should be scrutinized further prior to widespread use.     

Combination of ARDRA and RAPD genotyping techniques in identification of Acinetobacter spp. genomic species

A total of 10 non-repetitive multi-drug-resist-ant Acinetobacter strains were collected. With reference to A. calcoaceticus (ATCC23055), A. baumannii (ATCC19606), A. lwoffii (ATCC17986), and A. junii (NCTC5866), DNA fingerprint technique, amplified ribo-somal DNA restriction analysis (ARDRA), and random amplified polymorphism DNA (RAPD) were carried out to identify the genomic species of Acinetobacter spp. The distances between them were calculated by the unweighted pair group method with arithmetic (UPGMA). Genotypes ofAcinetobacter spp. were effectively classified and an A. junii together with nine A. baumannii isolates was genomically identified. The combination of ARDRA and RAPD DNA-fingerprint technique shows high com-plementarity, and could be a useful tool in Acinetobacter genomic species identification.     

Identification of New Delhi metallo-β-lactamase gene (NDM-1) from a clinical isolate of Acinetobacter junii in China

New Delhi metallo-β-lactamase-1 (NDM-1) is a novel type of metallo-β-lactamase (MBL) responsible for bacterial resistance to β-lactam antibiotics. Acinetobacter junii was previously shown to possess a MBL phenotype; however, the genes responsible for this phenotype were not identified. In this study, we reported the identification of NDM-1 gene in a clinical isolate of A. junii from a child patient in China, which was resistant to all β-lactams except aztreonam but sensitive to aminoglycosides and quinolones. The cloned NDM-1 gene contained an open reading frame of 813 bp and had a nucleotide sequence 99.9% identical (812/813) to reported NDM-1 genes carried by Acinetobacter baumannii , Enterococcus faecium , Escherichia coli , and Klebsiella pneumoniae . Recombinant NDM-1 protein was successfully expressed in E.?coli BL21, and antibiotic sensitivities of the NDM-1-producing E.?coli were largely similar to the A.?junii 1454 isolate. The findings of this study raise attention to the emergence and spread of NDM-1-carrying bacteria in China.