Legionella contamination of dental-unit waters.

Water samples collected from 28 dental facilities in six U.S. states were examined for the presence of Legionella pneumophila and other Legionella spp. by the PCR-gene probe, fluorescent-antibody microscopic, and viable-plate-count detection methods. The PCR and fluorescent-antibody detection methods, which detect both viable and viable nonculturable Legionella spp., gave higher counts and rates of detection than the plate count method. By the PCR-gene probe detection method, Legionella spp. were detected in 68% of the dental-unit water samples and L. pneumophila was detected in 8%. Concentrations of Legionella spp. in dental-unit water reached 1,000 organisms per ml or more in 36% of the samples, and 19% of the samples were in the category of 10,000/ml or above. L. pneumophila, when present in dental-unit water, never reached concentrations of 1,000/ml or more. Microscopic examination with fluorescent-antibody staining indicated that the contamination was in the dental-unit water lines rather than in the handpieces. Legionella spp. were present in 61% of potable water samples collected for comparative analysis from domestic and institutional faucets and drinking fountains; this percentage was not significantly different from the rate of detection of Legionella spp. in dental-unit water. However, in only 4% of the potable water samples did Legionella spp. reach concentrations of 1,000 organisms per ml, and none was in the 10,000 organisms-per-ml category, and so health-threatening levels of Legionella spp. in potable water were significantly lower than in dental-unit water. L. pneumophila was found in 2% of the potable water samples, but only at the lowest detectable level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct identification in food samples of Listeria spp. and Listeria monocytogenes by molecular methods

A new molecular approach for the detection and identification of Listeria spp. and Listeria monocytogenes in food is presented here. The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE). The protocol was first optimized by using strains from international collections. Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii. Moreover, for L. monocytogenes serotypes, partial differentiation was possible. The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment. Identification of 48 food isolates and direct detection of Listeria spp. in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp. and L. monocytogenes in food.     

铁线莲属植物的化学成分研究进展

多年来,铁线莲属许多植物的根作为传统中药“威灵仙”入药。近几十年来,从该属植物中分离得到多种化合物,如三萜皂苷、黄酮、生物碱、原白头翁素等成分。文章重点介绍了铁线莲属植物的化学成分研究进展,有助于对该属植物做进一步的开发。     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland.

The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% (Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% (L. monocytogenes 33.3%); this was higher than that in samples from dairy farms (Listeria spp. 8.8%; L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples (L. monocytogenes) and 1 of 33 soft cheese samples (L. seeligeri). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Development of liposome immunoassay for salmonella spp. using immunomagnetic separation and immunoliposome

The ability to detect Salmonella spp. is essential in the prevention of foodborne illness. This study examined a Salmonella spp. detection method involving the application of immunomagnetic separation and immunoliposomes (IMS/IL) encapsulating sulforhodamine B (SRB), a fluorescent dye. A quantitative assay was conducted by measuring the fluorescence intensity of SRB that was produced from an immunomagnetic bead-Salmonella spp.-immunoliposome complex. The results indicated detection limits of 2.7x10(5) and 5.2x10(3) CFU/ml for Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), respectivley. The signal/noise ratio was improved by using 4% skim milk as a wash solution rather than 2% BSA. In addition, higher fluorescence intensity was obtained by increasing the liposome size. Compared with the conventional plating method, which takes 3-4 days for the isolation and identification of Salmonella spp., the total assay time of 10 h only including 6 h of culture enrichment was necessary for the Salmonella detection by IMS/IL. These results indicate that the IMS/ IL has great potential as an alternative rapid method for Salmonella detection.     

Antibiotic resistance genes in multidrug-resistant Enterococcus spp. and Streptococcus spp. recovered from the indoor air of a large-scale swine-feeding operation

AIMS: In this study, multidrug-resistant bacteria previously recovered from the indoor air of a large-scale swine-feeding operation were tested for the presence of five macrolide, lincosamide and streptogramin (MLS) resistance genes and five tetracycline (tet) resistance genes. METHODS AND RESULTS: Enterococcus spp. (n = 16) and Streptococcus spp. (n =16) were analysed using DNA-DNA hybridization, polymerase chain reaction (PCR) and oligoprobing of PCR products. All isolates carried multiple MLS resistance genes, while 50% of the Enterococcus spp. and 44% of the Streptococcus spp. also carried multiple tet resistance genes. All Enterococcus spp. carried erm(A) and erm(B), 69% carried erm(F), 44% carried mef(A), 75% carried tet(M), 69% carried tet(L) and 19% carried tet(K). All Streptococcus spp. carried erm(B), 94% carried erm(F), 75% carried erm(A), 38% carried mef(A), 50% carried tet(M), 81% carried tet(L) and 13% carried tet(K). CONCLUSIONS: Multidrug resistance among airborne bacteria recovered from a swine operation is encoded by multiple MLS and tet resistance genes. These are the first data regarding resistance gene carriage among airborne bacteria from swine-feeding operations. SIGNIFICANCE AND IMPACT OF THE STUDY: The high prevalence of multiple resistance genes reported here suggests that airborne Gram-positive bacteria from swine operations may be important contributors to environmental reservoirs of resistance genes.     

电感耦合等离子质谱法(ICP-MS)法测定茵栀黄注射液中有害元素铅、砷、镉、汞、铜的含量

建立了电感耦合等离子体质谱法(ICP-MS)测定茵栀黄注射液中Pb、As、Cd、Hg、Cu 5种元素的方法。样品经微波消解后,直接用ICP-MS同时测定上述5种元素,结果5种元素的检出限分别在5～1250 ng/L之间;线性良好,线性相关系数均为r≥0.999;精密度RSD3.5%;回收率在95.7%～107.5%之间。方法操作简便、分析速度快、灵敏度高,各项分析性能指标均达到要求,适用于茵栀黄注射液中有害元素的测定。     

LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila

Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59% (98/107) and 93.33% (56/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100% (51/51) and 100% (18/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland

J. HARVEY AND A. GILMOUR. 1992. The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% ( Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% ( L. monocytogenes 33.3%); this was higher than that in samples from dairy farms ( Listeria spp. 8.8% L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples ( L. monocytogenes ) and 1 of 33 soft cheese samples ( L. seeligeri ). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Detection of Salmonella spp. and Listeria monocytogenes in suspended organic waste by nucleic acid extraction and PCR

A nucleic acid-based method for the detection of the bacterial pathogens Salmonella spp. and Listeria monocytogenes in biological waste was developed. The detection limits were less than 10 cells per ml of biological waste. The method does not include a phenol extraction step and can be easily performed in 1 to 2 days.     

Direct detection of Salmonella spp. in estuaries by using a DNA probe.

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.     

Lectotypification and description of Clematis viticella L. (Ranunculaceae)

The typification of Clematis viticella L. is discussed, and a lectotype designated from the Clifford Herbarium (BM). A standard specimen for C. viticella 'Purpurea Plena Elegans' has also been selected. Data from hybridization experiments and studies of natural distribution suggest that Clematis campaniflora Brotero is better treated as a subspecies, i.e. as C. viticella subsp. campaniflora (Brotero) Font Quer ex O. Bolòs & Vigo, than as either a species or variety.     

Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay

We report a duplex real-time PCR-based assay for the simultaneous quantitative detection of Listeria spp. and the food-borne pathogen Listeria monocytogenes. The targets of this single tube reaction were the 23S rDNA and hly genes of Listeria spp. and L. monocytogenes, respectively. Our assay was efficient, 100% selective (i.e., it allowed accurate simultaneous identification of 52 L. monocytogenes and 120 Listeria spp. strains through the FAM-labelled hly and the VIC-labelled 23S rDNA probes, respectively); and had a detection limit of one target molecule in 100% (23S rDNA) and 56% (hly) of the reactions. Simultaneous quantification was possible along a 5-log dynamic range, with an upper limit of 30 target molecules and R2 values > 0.995 in both cases. Our results indicate that this assay based on the amplification of the 23S rDNA gene can accurately quantify any mixture of Listeria species and simultaneously unambiguously quantify L. monocytogenes.     

Detection of Legionella spp. in cooling tower water by the polymerase chain reaction method.

The presence of Legionella spp. in cooling tower water was investigated by using the polymerase chain reaction. Total Legionella spp. detection was performed with 20-mer 5S rRNA complementary DNA sequence primers, and specific Legionella pneumophila detection was performed with 20-mer and then 21-mer macrophage infectivity potentiator gene sequence primers. Of 27 cooling tower water samples, 25 were positive for Legionella spp., and 14 of these contained L. pneumophila.     

铁线莲属灌木铁线莲组修订

对毛茛科铁线莲属Clematis的灌木铁线莲组sect. Fruticella进行了全面修订,确定此组共含5种、2 变种和3变型;对此组的分类学简史和地理分布做了介绍;写出本组2系的形态特征和地理分布,组下分类 群检索表,以及各种植物的形态描述、地理分布、生长环境等,并附有各分类群的3幅插图。根据花的构造 认为在铁线莲属中,灌木铁线莲组与黄花铁线莲组sect. Meclatis在亲缘关系上最为接近。二组拥有的共 同特征为: 花的4枚萼片呈黄色,斜上方开展;雄蕊花丝下部变宽,呈披针状条形。二组的区别为: 灌木铁 线莲组的萼片边缘在花开放后展宽成膜质狭翅,雄蕊无毛;而在黄花铁线莲组,萼片边缘不展宽,雄蕊花丝 被柔毛。本组5种的花构造一致;在其他营养器官形态特征方面观察到以下演化趋势: (1)叶由不分裂到羽 状全裂,由绿色到灰绿色,由狭菱状卵形到条形;(2)花序含数朵花,具花序梗和2苞片,生于当年生枝顶端, 到花数目减少到1朵,花序梗和苞片消失,花生于当年生枝的腋生短枝顶端。根据上述演化趋势认为灌木 铁线莲的模式变型C. fruticosa f. fruticosa (叶绿色,狭卵形或披针形;花序通常含数朵花,顶生于当年生枝 上)是本组的原始类型,其他种可能均源出于此类型。根据花序特征,本组被划分为2系。第1系,灌木铁线 莲系ser. Fruticosae,含4种,分布于我国黄土高原及相邻干旱地区,向北达蒙古戈壁荒漠,向南在甘肃越过 秦岭到达甘肃南部。在黄土高原北部及相邻的内蒙古西南部集中分布本系全部4种,这里是灌木铁线莲 组的分布中心。第2系,绿叶铁线莲系ser. Virides,含绿叶铁线莲C. viridis 1种,分布于我国横断山区北部的 高山灌丛草地。     

Legionella spp. in Puerto Rico cooling towers.

Water samples from air conditioning cooling towers receiving different treatment protocols on five large municipal buildings in San Juan, P.R., were assayed for various Legionella spp. and serogroups by using direct immunofluorescence. Several water quality parameters were also measured for each sample. Guinea pigs were inoculated with water samples to confirm pathogenicity and recover viable organisms. Legionella pneumophila serogroups 1 to 6, L. bozemanii, L. micdadei, L. dumoffii, and L. gormanii were observed in at least one of the cooling towers. L. pneumophila was the most abundant species; its density reached 10(5) cells per ml, which is within the range that is considered potentially pathogenic to humans. A significantly higher density of L. pneumophila was observed in the cooling tower water that was not being treated with biocides. Percent respiration (INT) and total cell activity (acridine orange direct count) were inversely correlated with bacterial density. This study demonstrates that Legionella spp. are present in tropical air-conditioning cooling systems and that, without continuous biocide treatment, they may reach densities that present a health risk.     

Detection of Salmonella spp. and Listeria monocytogenes in Suspended Organic Waste by Nucleic Acid Extraction and PCR

A nucleic acid-based method for the detection of the bacterial pathogens Salmonella spp. and Listeria monocytogenes in biological waste was developed. The detection limits were less than 10 cells per ml of biological waste. The method does not include a phenol extraction step and can be easily performed in 1 to 2 days.     

铁线莲属尾叶铁线莲组(毛茛科)基于形态学证据的分支系统学

选取铁线莲属 (Clematis) 尾叶铁线莲组(sect. Campanella)中37个种以及西南铁线莲组 (sect. Bebaeanthera) 中的2个种为内类群, 以Clematis alternata作为外类群, 通过对全世界10个标本馆的近2 000份腊叶标本的形态学特征统计, 选取了35个性状进行编码, 利用PAUP 4.0 beta 10 软件进行系统发育重建。通过最简约法(maximum parsimony)分析共得到182棵最简约分支树, 树长为182步, 一致性指数(CI)=0.385, 保持性指数(RI)=0.685。结果表明: (1)尾叶铁线莲组并非是一个单系类群; (2)以花序发生位置这一性状建立的sect. Bebaeanthera不能成立, 应并入尾叶铁线莲组; (3)本研究结果不支持 在尾叶铁线莲组中建立subsect. Henryianae或ser. Henryianae; (4) C. ranunculoides等萼片外面具纵翅的一群植物与本组中萼片红色的种类C. lasiandra和C. dasyandra有较近的亲缘关系; (5)C. otophora、C. pogonandra、C. repens和C.barbellata等几个种聚为一支, 且支持率很高, 它们具有一系列的共衍征, 即萼片质地较厚, 花丝扁平, 宽条形, 被短柔毛, 花药被黄色短毛, 药隔先端凸起, 因此不支持建立Ser. Pogonandrae; (6)本组中非洲分布的2个种无论从形态上还是从地理分布和生境上都十分特殊, 是本组植物的特化类群。     

Incidence of Listeria spp. in Breton live shellfish

Live shellfish samples (120) were collected from nine littoral sites in Brittany (western France). They were screened for Listeria spp. and a count of faecal coliforms was carried out. Analysis of the results revealed Listeria spp. in 55% of samples, a much higher rate than the previous, infrequent, recorded data. Furthermore, the study demonstrated that the frequency of Listeria spp. in winter was more important than in summer ( P < 0·001), and underlined a significant relationship between the occurrence of these bacteria and the concentration of faecal coliforms ( P < 0·001). Finally, comparison of the official and Gen-Probe^® methods revealed the limits of the standardized technique in the search for L. monocytogenes in shellfish.