Comparison of Detection Methods for Legionella Species in Environmental Water by Colony Isolation,Fluorescent Antibody Staining,and Polymerase Chain Reaction

Three detection methods for Legionella species in water samples from cooling towers and a river were examined. Direct counting of bacteria stained with fluorescent antibody (FA) for L. pneumophila (serogroups 1 to 6) could detect the cell of 10⁴ to 10⁶ cell/100 ml in all 14 samples, while colony counting method detected 10 to 10³ CFU/100 ml only in 8 samples from cooling towers. Polymerase chain reaction (PCR) assay with primers to amplify 16S ribosomal DNA sequence of most Legionella species (LEG primer) detected legionellae in 13 samples, while species-specific primers for L. pneumophila detected the DNAs from 3 samples. In laboratory examination, LEG primers could amplify DNAs of 29 species of genus Legionella with high sensitivity, even from 1 cell of L. pneumophila GIFU 9134. The PCR assay with LEG primers was specific and sensitive methods to be satisfied the survey of legionellae. Thus, PCR assay is a suitable method to detect and monitor Legionella species in an environment.     

Incidence of Legionella and heterotrophic bacteria in household rainwater tanks in Azumino,Nagano prefecture,Japan

Many administrative agencies in Japan are encouraging installation of household rainwater‐storage tanks for more effective use of natural rainwater. Water samples were collected periodically from 43 rainwater tanks from 40 households and tested for the presence of Legionella species and the extent of heterotrophic bacteria in Azumino city, Nagano prefecture, Japan. PCR assays indicated the presence of Legionella spp. in 12 (30%) of the 43 tank water samples. Attempts were made to identify correlations between PCR positive samples, topography, pH, chemical oxygen demand (COD), atmospheric temperature and the numbers of heterotrophic bacteria. Between June and October, 2012, the numbers of heterotrophic bacteria in rainwater tanks and the values of COD positively correlated with the presence of Legionella species. In most of the Legionella‐positive cases, heterotrophic bacterial cell counts were >10⁴ CFU/mL. Moreover, Legionella species were less frequently detected when the COD value was >5 mg KMnO₄/L. Therefore, at least in Azumino, Japan between June and October 2012, both heterotrophic bacterial counts and COD values may be considered index parameters for the presence of Legionella cells in rainwater tanks. Much more accumulation of such data is needed to verify the accuracy of these findings.     

<Emphasis Type="Italic">Amycolatopsis endophytica</Emphasis> sp. nov., a novel endophytic actinomycete isolated from oil-seed plant <Emphasis Type="Italic">Jatropha curcas</Emphasis> L.

A novel actinomycete, designated KLBMP 1221^T, was isolated from the surface-sterilized seeds of an oil-seed plant Jatropha curcas L. collected from Sichuan Province, south-west China and was characterized taxonomically by using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequence showed that this strain formed a distinct phyletic line within the radiation of the genus Amycolatopsis. The 16S rRNA gene sequence similarity indicated that strain KLBMP 1221^T was most closely related to Amycolatopsis eurytherma NT202^T (98.9%), Amycolatopsis tucumanensis ABO^T (98.8%), Amycolatopsis thermoflava N1165^T (98.6%) and Amycolatopsis methanolica IMSNU 20055^T (98.5%). Strain KLBMP 1221^T had morphological and chemotaxonomic properties that were consistent with its classification in the genus Amycolatopsis. However, DNA–DNA relatedness data and phenotypic differences clearly distinguished the isolate from its closest relatives. Based on the combined genotypic and phenotypic evidence, it is proposed that strain KLBMP 1221^T be classified as representative of a novel species for which the name Amycolatopsis endophytica sp. nov. is proposed. The type strain is KLBMP 1221^T (= KCTC 19776^T = CCTCC AA 2010003^T).     

Luteolibacter cuticulihirudinis sp. nov., isolated from Hirudo medicinalis

A Gram-negative, coccoid shaped bacterium isolated from the outer surface of the medicinal leech Hirudo medicinalis was characterized. The 16S rRNA gene sequence comparison revealed that the bacterium was closely related to species of the genus Luteolibacter. Luteolibacter pohnpeiensis was the most closely related species (94.6 % sequence similarity), followed by Luteolibacter luojiensis (93.4 %) and Luteolibacter algae (93.3 %). Chemotaxonomic data (major ubiquinone: MK-9; major polar lipids: phosphatidylethanolamine and phosphatidylglycerol; and major fatty acids: iso-C_14:0, C_16:0, iso-C_16:1, and anteiso-C_15:0) supported the affiliation of the isolate to the genus Luteolibacter. DNA–DNA hybridizations with the type strain of L. pohnpeiensis was 31 % (reciprocal value 30 %). A phenotypic differentiation of strain E100^T from L. pohnpeiensis and the other Luteolibacter species was possible by several physiological tests. We conclude Strain E100^T represents a novel species, for which we propose the name Luteolibacter cuticulihirudinis sp. nov. with the type strain E100^T (=CCM 8400^T = LMG 26924^T).     

<Emphasis Type="Italic">Pontibacter salisaro</Emphasis> sp. nov., Isolated from a clay tablet solar saltern in Korea

A Gram-negative, aerobic, rod-shaped, and red-pigmented bacterial strain, HMC5104^T, was isolated from a solar saltern, found in Jeungdo, Republic of Korea (34°59′47″N 126°10′02″E). The major fatty acids were summed feature 4 (comprising iso-C_17:1 I and/or anteiso-C_17:1 B; 37.2%), iso-C_15:0 (20.4%), and iso-C_17.0 30H (15.3%). The DNA G+C content was 46.0 mol%. The major isoprenoid quinone was menaquinone-7 (MK-7). A phylogenetic tree based on 16S rRNA gene sequences showed that strain HMC5104^T formed a lineage within the genus Pontibacter, and was closely related to Pontibacter korlensis (95.9%), P. roseus (94.9%), and P. actiniarum (94.3%). Similarities to all other Pontibacter species were between 95.9–93.9%. On the basis of the evidence presented in this study, strain HMC5104^T represents a novel species of the genus Pontibacter, for which the name Pontibacter salisaro sp. nov. is proposed. The type strain is HMC5104^T (=KCTC 22712^T = NBRC 105731^T).     

Arthrobacter wenxiniae sp. nov., a novel plant growth-promoting rhizobacteria species harbouring a carotenoids biosynthetic gene cluster

A bacterial strain, designated AETb3-4^T was isolated from the rhizosphere of lily. Comparison of 16S rRNA gene sequences showed that the sequence from strain AETb3-4^T exhibits high sequence similarity with those of Arthrobacter silviterrae KIS14-16^T (97.9%), Arthrobacter livingstonensis LI2^T (97.2%) and Arthrobacter stackebrandtii CCM 2783^T (97.0%). Whole genome average nucleotide identity (ANI) and the digital DNA-DNA hybridization (dDDH) values between strain AETb3-4T and the reference strains A. silviterrae DSM 27180^T, A. livingstonensis L12^T and A. stackebrandtii DSM 16005^T were below 83.6% and 27.7%, respectively, values which are considerably below the proposed thresholds for the species delineation, consistent with the proposal that strain AETb3-4^T represents a novel species. The genome size of strain AETb3-4^T is 4.33 Mb and the genomic DNA G?+?C content is 67.3%. The main polar lipids were identified as phosphatidylglycerol, diphosphatidylglycero, phosphatidylinositol and an unidentified glycolipid. The major fatty acids (>?10%) were identified as anteiso-C_{15: 0} and anteiso-C_{17: 0}. The predominant menaquinone was found to be menaquinone 9 (MK-9) (H₂) (82.2%). Phenotypic tests allowed the strain to be differentiated from its close phylogenetic neighbors. Based on the results obtained, it is proposed that the strain AETb3-4^T (=?CFCC 16390^T?=?LMG 31708^T) represents a novel species in the genus Arthrobacter, for which the names Arthrobacter wenxiniae sp. nov. is proposed. In addition, the novel strain AETb3-4^T has multiple plant growth-promoting characters including ACC-deaminase activity and production of IAA. Furthermore, the genome contains secondary metabolite biosynthesis gene clusters, including a carotenoid biosynthetic gene cluster, suggesting potential capacities for secondary metabolite synthesis. These data suggest that strain AETb3-4^T may have potential applications both in medicine and sustainable agriculture.

     

Nitratireductor lucknowense sp. nov., a novel bacterium isolated from a pesticide contaminated soil

A straw-yellow pigmented bacterium, strain IITR-21^T was isolated from a pesticide contaminated site and characterized by using a polyphasic taxonomic approach. The organism had morphological and chemotaxonomic properties consistent with its classification in the genus Nitratireductor. Phylogenetic analysis of the 16S rRNA gene sequence showed that the strain IITR-21^T belongs to the genus Nitratireductor and was moderately related to Nitratireductor indicus C115^T (97.7%) and Nitratireductor pacificus pht-3B^T (97.4%), whereas sequence similarity value with the other species including the type species of the genus Nitratireductor, Nitratireductor aquibiodomus showed less than 97.0% similarity. However, the DNA–DNA relatedness values between strain IITR-21^T and the moderately related taxa N. indicus (59.1%) and N. pacificus (40.4%) were well below the 70% threshold value recommended for the delineation of bacterial species. The G+C content of the DNA was 62.4 mol%. Based on physiological, biochemical tests and genotypic differences between the strain IITR-21^T and the other two validly published species of the genus Nitratireductor, it is proposed that the strain be classified as a new species of Nitratireductor as Nitratireductor lucknowense sp. nov. The type strain is IITR-21^T (=MTCC 8354^T = DSM 24322^T).     

Kibdelosporangium phytohabitans sp. nov., a novel endophytic actinomycete isolated from oil-seed plant Jatropha curcas L. containing 1-aminocyclopropane-1-carboxylic acid deaminase

A novel actinomycete, designated strain KLBMP 1111^T, was isolated from the root of the oil-seed plant Jatropha curcas L. collected from Sichuan Province, south-west China. Strain KLBMP 1111^T formed a distinct branch in the 16S rRNA gene phylogenetic tree together with the type strains in the genus Kibdelosporangium, with the highest similarity to Kibdelosporangium aridum subsp. aridum DSM 43828^T (98.8%), K. aridum subsp. largum DSM 44150^T (98.1%) and Kibdelosporangium philippinense DSM 44226^T (98.1%). The organism produced sporangium-like structures, the typical morphological characteristic of the genus Kibdelosporangium. The chemotaxonomic properties of this strain were also consistent with those of the genus Kibdelosporangium: the peptidoglycan contained meso-diaminopimelic acid; the predominant menaquinone was MK-9(H₄); phospholipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylinositol and an unknown phospholipid; iso-C_16:0, C_16:0, anteiso-C_15:0 and iso-C_15:0 as the predominant cellular fatty acids and the G+C content was 67.2 mol%. DNA–DNA hybridization values between strain KLBMP 1111^T and the three Kibdelosporangium species were less than 50%. This strain had the ability to produce a siderophore, utilized 1-aminocyclopropane-1-carboxylic acid (ACC) as sole source of nitrogen and possessed ACC deaminase enzyme. Based on genotypic and phenotypic data, strain KLBMP 1111^T represents a novel species in the genus Kibdelosporangium. We propose the name Kibdelosporangium phytohabitans sp. nov. for this species. The type strain is the strain KLBMP 1111^T (=KCTC 19775^T = CCTCC AA 2010001^T).     

<Emphasis Type="Italic">Thalassospira permensis</Emphasis> sp. nov., a new terrestrial halotolerant bacterium isolated from a naphthalene-utilizing microbial consortium

A halotolerant bacterium, strain SMB34^T, was isolated from a naphthalene-utilizing bacterial consortium obtained from primitive technogeneous soil (Verkhnekamsk salt deposit, Perm region, Russia) by enrichment procedure. The strain itself was unable to degrade naphthalene and grew at NaCl concentrations up to 11% (w/v). The 16S rRNA-based phylogenetic analysis showed that the strain belongs to the genus Thalassospira. The DNA-DNA hybridization values between SMB34^T and the type strains of phylogeneti-cally closest species (T. xiamenensis, T. profundimaris and T. tepidiphila) did not exceed 50%. The novel strain could be distinguished from the above species by the cell motility, MALDI/TOF mass spectra of whole cells and a range of physiological and biochemical characteristics. SMB34^T also considerably differs from the recently described species T. xianhensis, with the most striking differences in the DNA G + C content (53. ± 1.0 vs. 61.2 ± 1.0 mol %) and predominant ubiquinones (Q-10 vs. Q-9). The data obtained suggest strain SMB34^T (=VKM B-2527^T = NBRC 106175^T), designated as the type strain, represents a novel species, named Thalassospira permensis sp. nov.     

Pseudonocardia sichuanensis sp. nov., a novel endophytic actinomycete isolated from the root of Jatropha curcas L.

A novel isolate, designated strain KLBMP 1115^T was isolated from the surface-sterilized root of oil-seed plant Jatropha curcas L. collected from Sichuan Province, south-west China. Characterization of the isolate was based on a polyphasic approach. 16S rRNA gene sequence analysis indicated that strain KLBMP 1115^T belongs to the phylogenetic cluster of the genus Pseudonocardia and was most closely related to Pseudonocardia adelaidensis EUM 221^T (98.9%) and Pseudonocardia zijingensis DSM 44774^T (98.6%), whereas the DNA–DNA relatedness values between strain KLBMP 1115^T and the two type strains were 47.3 and 39.7%, respectively. Levels of lower similarities to the type strains of other recognized Pseudonocardia species ranged from 94.4 to 98.4%. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The predominant respiratory quinone was MK-8(H₄). The major fatty acids of strain KLBMP 1115^T was iso-C_16:0. The chemotaxonomic properties of strain KLBMP 1115^T were consistent with those shared by members of the genus Pseudonocardia. On the basis of the phenotypic features and the DNA–DNA hybridization data, strain KLBMP 1115^T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia sichuanensis sp. nov. is proposed. The type strain is KLBMP 1115^T (=KCTC 19781^T = CCTCC AA 2010002^T).     

Legionella clemsonensis sp. nov.: a green fluorescing Legionella strain from a patient with pneumonia

A novel Legionella species was identified based on sequencing, cellular fatty acid analysis, biochemical reactions, and biofilm characterization. Strain D5610 was originally isolated from the bronchial wash of a patient in Ohio, USA. The bacteria were gram‐negative, rod‐shaped, and exhibited green fluorescence under long wave UV light. Phylogenetic analysis and fatty acid composition revealed a distinct separation within the genus. The strain grows between 26–45°C and forms biofilms equivalent to L. pneumophila Philadelphia 1. These characteristics suggest that this isolate is a novel Legionella species, for which the name Legionella clemsonensis sp nov. is proposed.     

<Emphasis Type="Italic">Paenibacillus bryophyllum</Emphasis> sp. nov., a nitrogen-fixing species isolated from <Emphasis Type="Italic">Bryophyllum pinnatum</Emphasis>

A nitrogen-fixing, endospore-forming bacterium, designated strain L201^T was isolated from the leaves of Bryophyllum pinnatum growing in South China Agricultural University. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain L201^T is affiliated with the genus Paenibacillus, and closely related to Paenibacillus albidus Q4-3^T (97.4%), Paenibacillus odorifer DSM 15391^T (97.3%) and Paenibacillus borealis DSM 13188^T (97.2%). The main fatty acids components was anteiso-C_15:0 (48.1%). The predominant isoprenoid quinone was MK-7. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The G+C content of strain L201^T was 43.9%. DNA–DNA relatedness between L201^T and the reference strain was 29.8%. Biological and biochemical tests, protein patterns, genomic DNA fingerprinting and comparison of cellular fatty acids distinguished strain L201^T from the closely related Paenibacillus species. Based on these data, the novel species Paenibacillus bryophyllum sp. nov. is proposed, with the type strain L201^T(=?KCTC 33951 ^T?=?GDMCC 1.1251 ^T).     

<Emphasis Type="Italic">Pontibacter rhizosphera</Emphasis> sp. nov., isolated from rhizosphere soil of an Indian medicinal plant <Emphasis Type="Italic">Nerium indicum</Emphasis>

A gram-negative, motile, straight to curved rod shaped, pink pigmented bacterium was isolated from a soil sample collected from the rhizosphere of an Indian medicinal plant, Nerium indicum (Chuvanna arali) and subjected to a detailed polyphasic taxonomic study. The strain, designated as IMTB-1969^T, matched with most of the phenotypic and chemotaxonomic properties of the genus Pontibacter and represents a novel species. The major fatty acids of the strain were monounsaturated iso/anteiso branched C17 fatty acids (45.1%) and iso-C15:0 (16.5%). MK-7 was the predominant isoprenoid quinone. According to 16S rRNA gene sequence analysis, strain IMTB-1969^T was indicated to belonged to the phylum Bacteroidetes and further phylogenetic analysis revealed that the strain IMTB-1969^T belongs to the family Cytophagaceae and genus Pontibacter. The highest 16S rRNA gene sequence similarity was with Pontibacter korlensis CCTCC AB 206081^T (97.2%) and lower sequence similarity was observed with other species in the genus Pontibacter (95.9–94.0%). DNA–DNA relatedness study of the strain IMTB-1969^T confirmed that it represents a novel species. The G+C content of the genomic DNA was 52.2 (±0.5) mol%. The results of physiological and biochemical tests allowed the genotypic and phenotypic distinction of strain IMTB-1969^T from its closest phylogenetic relatives. The strain IMTB-1969^T should be classified as novel species of the genus Pontibacter, for which the name Pontibacter rhizosphera sp. nov. is proposed. The type strain is IMTB-1969^T (=MTCC 10673^T = DSM 24399^T).     

Detection of culturable and nonculturable Legionella species from hot water systems of public buildings in Japan

Aims: To investigate the prevalence of culturable and nonculturable Legionella species in hot water systems of public buildings in Japan and assess the risk factors associated with Legionella contamination in hot water systems. Methods and Results: Legionella species were detected by conventional culture and molecular methods in 130 water samples collected from 40 buildings. A total of 26 (20·0%) water samples from 17 (42·5%) buildings were positive by culture, qualitative PCR or both methods: Legionella pneumophila and Leg. anisa were detected in four samples by a culture method, whereas 23 samples were positive by qualitative PCR, with the presence of various Legionella species confirmed by sequencing. Of these 23 samples, bacterial counts were quantifiable in 21 by real‐time PCR (from 1·7 × 10⁵ to 2·6 × 10¹¹ cells per litre). Phylogenetic analysis of amplified partial 16S rRNA gene showed close relations to various species of Legionella, including Leg. anisa and Leg. micdadei, all of which have been associated with respiratory diseases or increased antibody titres in human sera. Assessment of risk factors showed that turbidity, free chlorine concentration, iron concentration and heterotrophic plate count (HPC) were significantly associated with Legionella contamination (P < 0·05). Conclusions: Contamination of hot water systems of public buildings with culturable and nonculturable Legionella species may be a potential risk factor for Legionella infection in Japan. Adequate levels of chlorine, low levels of iron and HPC are important maintenance measures in the reduction of Legionella contamination in hot water systems. Significance and Impact of the Study: More than 40% of hot water systems in the Japanese public buildings examined were contaminated by not only culturable Leg. pneumophila and Leg. anisa but also by nonculturable pathogenic species. To our knowledge, this is the first report of both culturable and nonculturable Legionella contamination in hot water systems of public buildings in Japan.     

Plasmid profiles ofLegionella species

Forty-six strains ofLegionella species were assayed for plasmid DNA content using routine laboratory procedures. Large-molecular-weight cryptic plasmids were detected inLegionella pneumophila serogroups 2, 3, and 4,L. bozemanii, L. dumoffii, L. micdadei, L. gormanii, L. longbeachae, and as yet unclassifiedLegionella-like organisms. No plasmids were found in strains ofL. pneumophila serogroups 1, 5, and 6. No correlations could be made between the possession of a specific plasmid profile, or lack of one, and any phenotypic markers such as virulence or antibiotic resistance. Several parameters were identified in this study as critical to the isolation of plasmid DNA fromLegionella: (i) DNA preparations obtained from frozen egg or animal materials had a higher incidence of detectable plasmid DNA than subcultures on bacteriologic media. (ii) A newly formulated broth supported exponential growth in all of the 46 strains; one strain required the addition of CO₂. (iii) Considerable heterogeneity was seen in cell susceptibility to various detergents. Since no single lytic agent was suitable for all strains, both ionic and noninic lysis methods were used with each strain. Within the limitations of both crude lysate preparations and the agarose gel electrophoresis method, this study identified a large 60–80 megadalton plasmid species in over 50% of the plasmid-containing strains.     

Presence of Legionella and Free-Living Amoebae in Composts and Bioaerosols from Composting Facilities

Several species of Legionella cause Legionnaires’ disease (LD). Infection may occur through inhalation of Legionella or amoebal vesicles. The reservoirs of Legionella are water, soil, potting soil and compost. Some species of free-living amoebae (FLA) that are naturally present in water and soil were described as hosts for Legionella. This study aimed to understand whether or not the composting facilities could be sources of community-acquired Legionella infections after development of bioaerosols containing Legionella or FLA. We looked for the presence of Legionella (by co-culture) and FLA (by culture) in composts and bioaerosols collected at four composting facilities located in southern Switzerland. We investigated the association between the presence of Legionella and compost and air parameters and presence of FLA. Legionella spp. (including L. pneumophila) were detected in 69.3% (61/88) of the composts and FLA (mainly Acanthamoeba, Vermamoeba, Naegleria and Stenamoeba) in 92.0% (81/88). L. pneumophila and L. bozemanii were most frequently isolated. FLA as potential host for Legionella spp. were isolated from 40.9% (36/88) of the composts in all facilities. In Legionella-positive samples the temperature of compost was significantly lower (P = 0.012) than in Legionella-negative samples. Of 47 bioaerosol samples, 19.1% (9/47) were positive for FLA and 10.6% (5/47) for L. pneumophila. Composts (62.8%) were positive for Legionella and FLA contemporaneously, but both microorganisms were never detected simultaneously in bioaerosols. Compost can release bioaerosol containing FLA or Legionella and could represent a source of infection of community-acquired Legionella infections for workers and nearby residents.     

<Emphasis Type="Italic">Pseudonocardia serianimatus</Emphasis> sp. nov., a novel actinomycete isolated from the surface-sterilized leaves of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

An aerobic, non-motile, catalase-positive, Gram-stain positive actinomycete designated YIM 63233^T was isolated from the surface-sterilized leaves of Artemisia annua L. and characterized using a polyphasic taxonomic approach. Optimal growth occurred at 20–28°C, pH 6.0–7.0 and in the presence of 0–3% (w/v) NaCl. 16S rRNA gene sequence-based phylogenetic analysis showed that strain YIM 63233^T clustered with species of the genus Pseudonocardia, displaying ≥1.2% sequence divergence with recognized species of this genus (from 98.8 to 94.0%). Relatively low levels of DNA–DNA relatedness were found between strain YIM 63233^T and Pseudonocardia petroleophila IMSNU 22072^T, which supported the classification of strain YIM 63233^T within a novel species of the genus Pseudonocardia. The G + C content of genomic DNA was 72.0 mol%. Strain YIM 63233^T possessed chemotaxonomic markers that were consistent with classification in the genus Pseudonocardia, i.e. the predominant fatty acids were iso-C16:0 (32.27%), C16:0 10-methyl (8.73%) and C17:1ω8c (8.30%), whilst the predominant menaquinone was MK-8(H₄). The diagnostic diamino acid of the cell-wall peptidoglycan was meso-diaminopimelic acid. The major cell wall sugars were glucose, arabinose, galactose, mannose and rhamnose. The results of physiological and biochemical tests and DNA–DNA hybridization allowed the phenotypic and genotypic differentiation of strain YIM 63233^T from its closest phylogenetic neighbours. Therefore, the new isolate YIM 63233^T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia serianimatus sp. nov. is proposed. The type strain is YIM 63233^T (=DSM 45302^T = CCTCC AA 208079^T).     

Usefulness of real-time PCR as a complementary tool to the monitoring of Legionella spp. and Legionella pneumophila by culture in industrial cooling systems

Aims: This study was designed to evaluate the usefulness of quantification by real‐time PCR as a management tool to monitor concentrations of Legionella spp. and Legionella pneumophila in industrial cooling systems and its ability to anticipate culture trends by the French standard method (AFNOR T90‐431). Methods and Results: Quantifications of Legionella bacteria were achieved by both methods on samples from nine cooling systems with different water qualities. Proportion of positive samples for L. pneumophila quantified by PCR was clearly lower in deionized or river waters submitted to a biocide treatment than in raw river waters, while positive samples for Legionella spp. were quantified for almost all the samples. For some samples containing PCR inhibitors, high quantification limits (up to 4·80 × 10⁵ GU l^?1) did not allow us to quantify L. pneumophila, when they were quantified by culture. Finally, the monitoring of concentrations of L. pneumophila by both methods showed similar trends for 57–100% of the samples. Conclusions: These results suggest that, if some methodological steps designed to reduce inhibitory problems and thus decrease the quantification limits, could be developed to quantify Legionella in complex waters, the real‐time PCR could be a valuable complementary tool to monitor the evolution of L. pneumophila concentrations. Significance and Impact of the Study: This study shows the possibility of using real‐time PCR to monitor L. pneumophila proliferations in cooling systems and the importance to adapt nucleic acid extraction and purification protocols to raw waters.     

<Emphasis Type="Italic">Pseudonocardia bannaensis</Emphasis> sp. nov., a novel actinomycete isolated from the surface-sterilized roots of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

During the course of our research on new actinobacterial sources, a novel actinomycete strain YIM 63101^T was isolated from the surface-sterilized roots of Artemisia annua L. collected from Xishuangbanna, Yunnan province, south-west China and characterized by using a polyphasic approach. The strain formed well-differentiated aerial and substrate mycelia. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 63101^T belongs to the genus Pseudonocardia, with highest similarity to “Pseudonocardia artemisiae YIM 63587^T” (99.4%). Sequence similarities between strain YIM 63101^T and the other Pseudonocardia species ranged from 97.0 (Pseudonocardia saturnea IMSNU 20052^T) to 94.0% (Pseudonocardia compacta IMSNU 20111^T). The chemotaxonomic characteristics, such as cell wall diaminopimelic acid, whole-cell sugars, fatty acid components and the major menaquinones suggested that the organism belonged to the genus Pseudonocardia. The G + C content of the genomic DNA was 69.4 mol%. Based on comparative analysis of physiological, biochemical and chemotaxonomic data, including low DNA–DNA hybridization results, it is proposed that strain YIM 63101^T represents a novel species of the genus Pseudonocardia, named Pseudonocardia bannaensis sp. nov. The type strain is YIM 63101^T (= CCTCC AA 208077 ^T = DSM 45300^T).     

<Emphasis Type="Italic">Actinopolyspora xinjiangensis</Emphasis> sp. nov., a novel exteremely halophilic actinomycete isolated from a salt lake in Xinjiang,China

A novel halophilic, filamentous actinobacterium, designated TRM 40136^T, was isolated from a hypersaline habitat in Xinjiang Province, north-west China. The strain is aerobic, Gram-positive, halophilic, and the optimum NaCl concentration for growth is 10–15% (w/v). The whole-cell sugar pattern consists of xylose, glucose and arabinose. The predominant menaquinone is MK-6 (51.2%) and the major fatty acids are anteiso-C15:0 (35.2%), anteiso-C17:0 (15.9%) and iso-C15:0 (13.7%). The phospholipid pattern consists of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and two unknown phospholipids. The G + C content of the genomic DNA is 68.9 mol%. Phylogenetic analysis showed that strain TRM 40136^T had 16S rRNA gene sequence similarity of 96.1% with the closest described species Actinopolyspora mortivallis, and it can be distinguished from all species in the genus Actinopolyspora by using these data of polyphasic taxonomy study. On the basis of the polyphasic evidence, the strain TRM 40136^T should be designated as a novel species of the genus Actinopolyspora for which the name Actinopolyspora xinjiangensis sp.nov. is proposed. The type strain is TRM 40136^T (=CCTCC AA 209080^T = KCTC 19656^T).