16S-23S rDNA Intergenic Spacer Research Articles

Halogenase Genes in Nonribosomal Peptide Synthetase Gene Clusters of Microcystis (Cyanobacteria): Sporadic Distribution and Evolution

Cyanobacteria of the genus Microcystis are known to produce secondary metabolites of large structural diversity by nonribosomal peptide synthetase (NRPS) pathways. For a number of such compounds, halogenated congeners have been reported along with nonhalogenated ones. In the present study, chlorinated cyanopeptolin- and/or aeruginosin-type peptides were detected by mass spectrometry in 17 out of 28 axenic strains of Microcystis. In these strains, a halogenase gene was identified between 2 genes coding for NRPS modules in respective gene clusters, whereas it was consistently absent when the strains produced only nonchlorinated corresponding congeners. Nucleotide sequences were obtained for 12 complete halogenase genes and 14 intermodule regions of gene clusters lacking a halogenase gene or containing only fragments of it. When a halogenase gene was found absent, a specific, identical excision pattern was observed for both synthetase gene clusters in most strains. A phylogenetic analysis including other bacterial halogenases showed that the NRPS-related halogenases of Microcystis form a monophyletic group divided into 2 subgroups, corresponding to either the cyanopeptolin or the aeruginosin peptide synthetases. The distribution of these peptide synthetase gene clusters, among the tested Microcystis strains, was found in relative agreement with their phylogeny reconstructed from 16S–23S rDNA intergenic spacer sequences, whereas the distribution of the associated halogenase genes appears to be sporadic. The presented data suggest that in cyanobacteria these prevalent halogenase genes originated from an ancient horizontal gene transfer followed by duplication in the cyanobacterial lineage. We propose an evolutionary scenario implying repeated gene losses to explain the distribution of halogenase genes in 2 NRPS gene clusters that subsequently defines the seemingly erratic production of halogenated and nonhalogenated aeruginosins and cyanopeptolins among Microcystis strains.

Rapid investigation of French sourdough microbiota by restriction fragment length polymorphism of the 16S-23S rRNA gene intergenic spacer region

The aim of the present research is to identify rapidly the lactic acid bacteria (LAB) microflora of four natural French sourdoughs (GO, BF, VB and RF), applying a biphasic (restriction length polymorphism (RFLP) and sequencing) approach for bacterial identification. For this purpose, a database with the RFLP patterns of 30 lacto- bacilli type strains was created. So-developed ISR-RFLP algorithm was further applied for the differentiation and identification of 134 sourdough isolates. The 16S-23S rDNA intergenic spacer region was amplified by primers t Ala and 23S/10, and then digested by HindIII, HinfI and a-TaqI enzymes. Nucleotide sequences of the cloned 16S- 23S intergenic spacer region (ISR) were determined by the dideoxynucleotide chain termination method. The T7Prom and M13rev primers flanking the multiple cloning site of pCR2.1 DNA were used to sequence both DNA strands. The RFLP profile obtained upon digestion with HindIII, HinfI and a-TaqI enzymes can be used to discriminate Lactobacillus sanfranciscensis (66%), Lactobacillus panis (17%), Lactobacillus nantensis (11%) and Lactobacillus hammesii(6%) in sourdough GO, Lactobacillus sanfran- ciscensis (80%), Lactobacillus spicheri (14%) and Lactobacillus pontis(6%) in sourdoughs BF. In sourdoughs VB, which differed in the process temperature, we can differentiate Lactobacillus sanfranciscensis (89%) and Leuconostoc mesenteroidessubsp. mesenteroides (11%). Lactobacillus frumenti(47%), Lactobacillus hammesii (8%), and Lactobacillus paralimentarius (45%) were dif- ferentiated in sourdough RF.

Molecular identification of Arcanobacterium bialowiezense and Arcanobacterium bonasi based on 16S–23S rRNA intergenic spacer region sequences

In the present study, the 16S–23S rDNA intergenic spacer region (ISR) of Arcanobacterium ( A.) bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483 and A. pyogenes DSM 20630 was amplified, sequenced and compared with the corresponding 16S rRNA gene sequences yielding comparable phylogenetic relationships. The ISR sequence of A. bialowiezense and A. bonasi allowed the design of species-specific oligonucleotide primers which could successfully be used for PCR-mediated identification of previously characterized A. bialowiezense and A. bonasi isolated from infections of the European bison. The presented molecular identification might help to improve a future diagnosis of both newly described bacterial pathogens.

Characterization of Dickeya strains isolated from potato and river water samples in Finland

Plant pathogenic enterobacteria in the genera Pectobacterium and Dickeya (formerly classified as Erwinia) were isolated from diseased potato stems and tubers. The isolated bacteria were identified as P. atrosepticum, P. carotovorum and pathogens in the genus Dickeya with PCR tests. Furthermore, Dickeya strains were isolated from river water samples throughout the country. Phylogenetic analysis with 16S-23S rDNA intergenic spacer sequences suggested that the Dickeya strains could be divided into three groups, two of which were isolated from potato samples. Phylogenetic analysis with 16S rDNA sequences and growth at 39°C suggested that one of the groups corresponds to D. dianthicola, a quarantine pathogen in greenhouse cultivation of ornamentals, while two of the groups did not clearly resemble any of the previously characterised Dickeya species. Field trials with the strains indicated that D. dianthicola-like strains isolated from river samples caused the highest incidence of rotting and necrosis of potato stems, but some of the Dickeya strains isolated from potato samples also caused symptoms. The results showed that although P. atrosepticum is still the major cause of blackleg in Finland, virulent Dickeya strains were commonly present in potato stocks and rivers. This is the first report suggesting that Dickeya, originally known as a pathogen in tropical and warm climates, may cause diseases in potato in northern Europe.

Bartonella australissp. nov. from Kangaroos, Australia

To the Editor: During April–May 1999, 3 Bartonella isolates (AUST/NH1, AUST/NH2, AUST/NH3) were cultivated and established from the blood of 5 Macropus giganteus gray kangaroos from central coastal Queensland, Australia. We used multigene sequencing to evaluate whether these Bartonella isolates fulfill the minimum requirements for classification as a new species. DNA from each Bartonella isolate was extracted by using the QIAamp tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Partial PCR amplification and sequencing of the genes encoding the 16S rDNA (rrs), citrate synthase (gltA), β-subunit of the RNA polymerase (rpoB), and cell division protein (ftsZ), as well as for the 16S–23S rDNA intergenic spacer (ITS) were attempted by using previously described primers and conditions (1). Bartonella sp. isolates AUST/NH1 to AUST/NH3 exhibited identical sequences for all 4 genes and the spacer studied, and isolate AUST/NH1 was selected as type strain among kangaroo isolates. Similarity rates between strain Aust/NH1 and validated Bartonella species (Appendix Table) ranged from 84.7% to 91.6%, from 97.5% to 98.5%, from 79.6% to 87.2%, from 85.4% to 95.0%, and from 83.5% to 87.1% for the ITS and rrs, gltA, rpoB, and ftsZ genes, respectively. Therefore, for each of these 4 genes or the spacer, strain AUST/NH1 exhibited similarity rates with all other species lower than the cutoffs published to classify Bartonella isolates within a validated species (1). It may thus be regarded as a new species. To estimate the genomic G+C content of strain AUST/NH1, we amplified and sequenced its ftsY gene as described (2) by using the BartftsyF (5′-ATGACAAAAYCYTTTATMAA-3′) and BartftsyR (5′-TCATGAGTGTCTTCCTGC-3′) primers. The ftsY G+C content was 37.7%; the calculated genomic G+C content was 39.51%. The ftsY sequence was deposited in GenBank under accession no. {type:entrez-nucleotide,attrs:{text:DQ538398,term_id:109649511,term_text:DQ538398}}DQ538398. The phylogenetic relationships among the studied bartonellae were inferred from sequence alignments of each gene and from concatenated gene sequences by using the maximum parsimony and neighbor-joining methods within the MEGA version 2.1 software package (3) and the maximum-likelihood method within the PHYLIP software package (4). Using rrs, gltA, and rpoB sequences, the phylogenetic position of strain AUST/NH1 was supported by bootstrap values <70%. In contrast, by using the ITS, ftsZ, and concatenated sequences, strain AUST/NH1 clustered with a group of B. tribocorum, B. grahamii, and B. elizabethae, with elevated bootstrap values according to the 3 analysis methods (Figure). Figure Unrooted dendrogram showing the phylogenetic position of Bartonella sp. strain AUST/NH1 among Bartonella species inferred from the comparison of concatenated sequences from the rrs, gltA, intergenic spacer, rpoB, and ftsZ genes by the neighbor-joining ... The Bartonella strains we describe are the first, to our knowledge, obtained from kangaroos and, more generally, from marsupials. Before this study, the only 2 Bartonella species found in Australia were B. henselae (5) and B. quintana (6). We demonstrated that strain AUST/NH1 was reliably associated with a well-established cluster, including the rodent-associated B. elizabethae, B. grahamii, and B. tribocorum (7). Therefore, we are confident that the phylogenic position of the new Bartonella, which was similar according to 3 analysis methods and supported by high bootstrap values, is reliable. Although B. grahamii (8) and B. elizabethae (9), members of the same phylogenetic cluster as strain AUST/NH1, cause human infections, the pathogenicity of B. tribocorum is as yet unknown. Its pathogenicity should therefore be investigated, especially for persons who come in contact with kangaroos. B. australis is a facultative intracellular gram-negative bacterium. It grows on Columbia agar with 5% sheep blood at 32°C to 37°C in a moist atmosphere containing 5% CO2. A primary culture was obtained after 7 days, and subculture was obtained after 4 days under the same conditions. Colonies are homogeneous, smooth, round, and gray-white. The 3 strains tested were oxidase negative, catalase negative, and nonmotile. Pathogenicity for humans is, as yet, unknown. The type strain is strain AUST/NH1. The new species is distinguished from other Bartonella species by its 16S rRNA, gltA, rpoB, ftsZ gene sequences, as well as its 16S–23S rRNA ITS sequence. The estimated G+C content is 38%. The type strain exhibits a specific serotype (10) and was susceptible to amoxicillin, ceftriaxone, imipenem, erythromycin, clarithromycin, ofloxacin, ciprofloxacin, rifampin, and tetracycline (unpub. data). The type strain AUST/NH1 has been deposited in the Collection of the World Health Organization Collaborative Center for Rickettsioses, Borrelioses and Tick-borne Infections (CSUR), Marseille, France, under reference CSUR B1; in the Collection de l’Institut Pasteur (CIP) under reference CIP 108978T; and in the Culture Collection of the University of Goteborg (CCUG), Sweden, under reference CCUG 51999. The strains AUST/NH2 and AUST/NH3 have been deposited in CSUR under references CSUR B2 and CSUR B3, in the CIP under references CIP 108980 and CIP 108979, and in CCUG under references CCUG 52000 and CCUG 52001, respectively.

Assessment of the 16S‐23S rDNA Intergenic Spacer Region in Enterococcus spp. for Microbial Source Tracking

A new library-based microbial source tracking (MST) approach intended for initial application in the coastal waters of Virginia was evaluated. Host-origin isolates of Enterococcus spp. were collected from beaches and the surrounding tidewater region of Virginia and used to construct a library based on the pattern of DNA band lengths produced by the amplification of the 16S-23S rDNA intergenic spacer (IGS) region, and subsequent digestion with MboI. Initial results from small host-origin libraries (64 and 200 total isolates) with discriminant analysis (DA) and logistic regression (LR) yielded high average rates of correct classification (ARCC) for a four-source classification split (birds, dogs, sewage, and wildlife), with ARCCs ranging from 83 to 100%. However, the poor results obtained when classification was attempted on a non-library validation set (VS, ARCCs of 47 and 48%, respectively, using DA and LR) demonstrated that a library of 200 isolates was insufficient to adequately represent the diversity of the enterococci in the sampled region. An increase in the library size to 1029 total isolates was accompanied by a reduction in the ARCC of the library to 42.7% with DA and 45.7% with LR, plus similarly poor results obtained from the VS. The low correct classification rates generated by the larger known-source library were unsuitable for field application. Many reported MST methods have been based on results obtained using small host-origin libraries without external validation. Our results indicate that such an approach can be very misleading, and that larger libraries and external validation is essential for the confirmation of preliminary results.

High inter-species and low intra-species variation in 16S–23S rDNA spacer sequences of pathogenic avian mycoplasmas offers potential use as a diagnostic tool

In order to investigate its value for phylogenetic analysis, species characterisation and diagnosis, the 16S–23S rDNA intergenic spacer regions (ISRs) of the type strain of 23 avian Mycoplasma species were amplified and the sequences determined. Also sequenced were the reference strains of Mycoplasma iowae serotypes J, K, N, Q and R and a number of field strains of Mycoplasma synoviae, Mycoplasma gallisepticum, Mycoplasma meleagridis and M. iowae. The ISRs demonstrated a high level of size variation (178–2488 bp) between species and did not include tRNA genes. Phylogenetic analysis performed using the information conflicted with that based on the 16S rDNA and was therefore not helpful for phylogenetic studies. However, the ISR did appear to be of value for determining species since there was high inter-species variation between all 23 avian Mycoplasma species, and in addition there was low intra-species variation, at least in the four pathogenic species. It could also be very useful as additional information in the description of a new species and as a target for species-specific PCRs.

Different Mesorhizobium species sharing the same symbiotic genes nodulate the shrub legume Anagyris latifolia

The isolation and characterization of six rhizobial strains isolated from Anagyris latifolia, a shrub legume endemic to the Canary Islands, is reported in this study. The isolates were characterized by 16S-ARDRA, and sequencing of the ribosomal 16S rRNA gene, the 16S–23S rDNA intergenic spacer region, and the housekeeping gene for glutamine synthetase II ( glnII). The phylogenies based on the three types of sequences matched, showing that the isolates belonged to three distinct lineages within the genus Mesorhizobium that could represent different species. However, the ribosomal and glnII phylogenies revealed some discrepancies in the relationships between the isolates and the named species in this genus. Despite their different taxonomic affiliation, all the isolates showed identical nodC sequences which were closely related (95% similarity) to that of the Mesorhizobium tianshanense type strain, indicating that they must have acquired the nodulation genes by a phenomenon of lateral gene transfer.

Phylogenetic Relationships of Xylella fastidiosa Strains Isolated from Landscape Ornamentals in Southern California

ABSTRACT Xylella fastidiosa is an insect-borne, xylem-limited pathogenic bacterium that has been associated with a rise in incidence of diseased landscape ornamentals in southern California. The objective of this study was to genetically characterize strains isolated from ornamental hosts to understand their distribution and identity. Strains of X. fastidiosa isolated from ornamentals were characterized using a multiprimer polymerase chain reaction (PCR) system, random amplified polymorphic DNA (RAPD)-PCR, and sequence analysis of the 16S-23S rDNA intergenic spacer region (ISR). Based on RAPD-PCR and 16S-23S rDNA ISR, strains isolated from daylily, jacaranda, and magnolia clustered with members of X. fastidiosa subsp. sandyi and caused oleander leaf scorch but not Pierce's disease symptoms in glasshouse assays on oleander and grape, respectively. This demonstrated both that our groupings based on genetic characterization were valid and that strains of X. fastidiosa subsp. sandyi are present in hosts other than oleander. Strains isolated from Spanish broom, cherry, and one strain isolated from western redbud clustered with X. fastidiosa subsp. fastidiosa members. Strains isolated from purple-leafed plum, olive, peach, plum, sweetgum, maidenhair tree, crape myrtle, and another western redbud strain clustered with members of X. fastidiosa subsp. multiplex. All strains isolated from mulberry and one from heavenly bamboo formed a separate cluster that has not yet been defined as a subspecies.

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S?23S rDNA intergenic spacer

To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.

PCR monitoring of Lactobacillus and Bifidobacterium dynamics in fermentations by piglet intestinal microbiota

A new group-specific primer (Lact71R), targeting the 16S-23S rDNA intergenic spacer region of Lactobacillus, was tested in its specificity to amplify rDNA of lactobacilli from piglet intestinal origin by polymerase chain reaction (PCR). Lact71R and Lab0677F, a Lactobacillus group-specific primer targeting the 16S rDNA, generated a common amplicon by PCR with DNA from Lactobacillus and Pediococcus reference strains, but not from Weissella strains. Sequence analysis of clones obtained by PCR amplification with Lact71R and Lab0677F and total DNA isolated from the ileal, caecal and colonic contents of one piglet resulted in Lactobacillus and Lactobacillus-like sequences mainly retrieved from intestinal environments. The primer pair was further validated in a culture independent PCR-analysis to monitor broad fluctuations of lactobacilli populations in fructo-oligosaccharides (FOS) fermentations by piglet intestinal microbiota. Bifidobacterium genus-specific primers were also used for PCR titre determination throughout FOS fermentations, in parallel with lactate and short chain fatty acids (SCFA) quantification. Increases between PCR titres were correlated with lactate detection in early stages of fermentation. Based on the obtained results, a simple monitoring PCR approach is proposed, foreseeing its application to the study of the dynamics of specific bacterial populations in complex environments.

P588 Phenotypic versus genotypic identification of bacteria in the clinical microbiology laboratory: the possible impact on patient care

229 strains of Staphylococcus isolated from the intestines of wild small mammals (Insectivora and rodents) were analysed by phenotypic tests and 16S–23S rDNA intergenic spacer PCR amplification (ITS-PCR). Based on the results of three methods (phenotypical tests, API numerical profiles and ITS-PCR) 65.5% of all staphylococcal strains could be identified. The remaining strains were compared by cluster analysis with reference and identified strains. It is quite possible that, a part of unidentified strains represent hitherto undescribed species, all the more as their ITS types were not found among other members of the genus Staphylococcus. Moreover, the results show that small mammals provides a suitable new habitat for certain staphylococci.

Detection and identification of the phytoplasma associated with pear decline in Taiwan

Pear decline (PD) is an important phytoplasmal disease that occurs mainly in Europe and North America. In 1994, pear trees exhibiting symptoms typical of PD disease were observed in orchards of central Taiwan. The sequence of 16S rDNA and 16S–23S rDNA intergenic spacer region (ISR) of the causative agent of pear decline in Taiwan (PDTW) were amplified with polymerase chain reaction (PCR) using a DNA template prepared from the diseased leaves. Sequence analysis of 16S rDNA revealed that the PDTW agent was closely related to the phytoplasmas of the apple proliferation group that cause diseases in stone fruits, pear and apple. Consistent with the result of 16S rDNA sequence analysis, sequence analysis of the 16S–23S rDNA ISR and putative restriction site analyses of 16S rDNA and 16S–23S rDNA ISR sequences provided further support for the view that the PDTW phytoplasma causing pear decline in Taiwan may represent a new subgroup of the apple proliferation group. According to the rDNA sequence of PDTW phytoplasma, two specific PCR primer pairs, APf2/L1n and fPD1/rPDS1, were designed in this study for the detection of the etiological agent in pear trees and insect vectors. Based on the sequence analyses of the PCR-amplified fragments, two species of pear psyllas, Cacopsylla qianli and Cacopsylla chinensis, were found to carry PDTW phytoplasma.

The rrn locus and gyrB genotyping confirm the existence of two clonal groups in strains of Yersinia enterocolitica subspecies palearctica biovar 1A

Eighty-one strains of Yersinia enterocolitica biovar 1A representing several serotypes isolated from India, France, Germany and the USA were analyzed using ribotyping, 16S–23S rDNA intergenic spacer length polymorphism analysis (PCR-ribotyping) and gyrB restriction fragment length polymorphism. Ribotyping with BglI, NciI and EcoRV distinguished 81 strains into 4, 3 and 2 ribotypes respectively. BglI- NciI combination gave the highest Simpson's diversity index (DI = 0.43). Strains with identical ribotypes were further differentiated by PCR-ribotyping. The combination of BglI- NciI ribotyping with PCR ribotyping increased DI to 0.72. This suggested that the combination of the two may be used for molecular epidemiological studies of Y. enterocolitica biovar 1A. This approach clearly resolved the strains into two clonal groups, each comprising strains isolated from humans, swine, pork and wastewater. PCR-RFLP of the gyrB gene using three enzymes ( AluI, MspI and HinfI) distinguished strains into seven types and confirmed the existence of two clonal groups. Thus, assessment of heterogeneity based on chromosomal restriction analysis (ribotyping), rRNA spacer length polymorphism (PCR-ribotyping) and gyrB gene analysis were in concordance and provided unequivocal evidence for the presence of two groups amongst strains of Y. enterocolitica biovar 1A despite their diverse geographic origins. These data also grouped clinical and non-clinical strains of serotype O:6,30–6,31 into discrete subgroups.

Efficacy of recA gene sequence analysis in the identification and discrimination of Lactobacillus hilgardii strains isolated from stuck wine fermentations

Conventional phenotypic methods sometimes lead to misidentification of some heterofermentative wine lactobacilli such as Lactobacillus hilgardii, Lactobacillus buchneri, and Lactobacillus brevis. We establish the specificity of 16S rDNA sequencing in the differentiation of these species and in the rejection of the Lactobacillus vermiforme species name. Moreover, we succeeded in differentiating these heterofermentative species by means of recA gene sequence comparison. Short homologous regions were amplified by PCR with degenerate consensus primers, sequenced, and 280 bp were analysed and considered for the inference of phylogenetic trees. The phylogram obtained was coherent and clearly separated the three species. The recA gene sequence was a reliable and useful method that allowed a good discrimination among closely related species. The validity of the recA gene sequence, restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S–23S rDNA intergenic spacer region (ISR), and random amplified polymorphic DNA (RAPD) to study the L. hilgardii intraspecies heterogeneity was tested in five strains isolated from stuck wine fermentations at the same winery in the same vintage. The results indicated that L. hilgardii is a heterogeneous species. Since L. hilgardii is a malolactic species that can influence the final quality of the wine, the presence of oenological relevant genes, such as those involved in ethyl carbamate or biogenic amine production, was investigated.

Genetic diversity of rhizobia associated with Desmodium species grown in China

Desmodia are leguminous plants used as important forage and herbal medicine in China. Little information is available about the nodule bacteria of Desmodium species. To understand the genetic diversity of rhizobia associated with Desmodium species grown in China, isolates from temperate and subtropical regions were obtained and analysed. A total of 39 rhizobial strains isolated from 9 Desmodium species grown in China were characterized by PCR-based 16S rDNA gene and 16S-23S rDNA intergenic spacer gene restriction fragment length polymorphism (RFLP) and 16S rRNA gene sequencing. The results showed high diversity among rhizobia symbiotic with Desmodium species. Most microsymbionts of Desmodium species belonged to Bradyrhizobium closely related to Bradyrhizobium elkanii, Bradyrhizobium japonicum and Bradyrhizobium yuanmingense. Several small groups or single strain were related to Rhizobium, Sinorhizobium or Mesorhizobium. Desmodium species formed nodules with diverse rhizobia in Chinese soils. These results offered the first systematic information about the microsymbionts of desmodia grown in the temperate and subtropical regions of China.

Relatedness of Streptococcus equi subsp. zooepidemicus strains isolated from harbour seals ( Phoca vitulina) and grey seals ( Halichoerus grypus) of various origins of the North Sea during 1988–2005

The present study was designed to identify 15 beta-hemolytic streptococci isolated during a period between 1988 and 2005 from nine harbour seals and six grey seals from various origins of the North Sea. All isolates were identified as Streptococcus equi subsp. zooepidemicus. The bacteria were additionally investigated for relatedness by restriction fragment length polymorphism analysis of PCR amplified 16S-23S rDNA intergenic spacer region and gene szp and by macrorestriction analysis of chromosomal DNA of the strains by pulsed field gel electrophoresis. The molecular analysis yielded identical or closely related patterns within the strains of the present study and with the S. equi subsp. zooepidemicus strains isolated from harbour seals of German North Sea which were investigated previously [Akineden, Ö., Hassan, A.A., Alber, J., El-Sayed, A., Estoepangestie, A.T.S., Lämmler, C., Weiss, R., Siebert, U., 2005. Phenotypic and genotypic properties of S. equi subsp. zooepidemicus isolated from harbor seals ( Phoca vitulina) from the German North Sea during the phocine distemper outbreak in 2002. Vet. Microbiol. 110, 147–152]. This indicates that this single or closely related bacterial clone existed during both phocine distemper virus epidemics in 1988 and 2002 and that a direct transmission of the strains has occurred between two seal species and between seal populations of far distant regions possibly with grey seals as a vector.

Identification of the Bacterial Leaf Spot Pathogen of Poinsettia in China

AbstractBacterial leaf spot of poinsettia has been first reported in China in 2003 and ascribed to Xanthomonas campestris pv. poinsettiicola following the traditional classification of xanthomonads. The present study was undertaken to elucidate the precise position of the causal bacterium in the newly proposed classification of xanthomonads, three bacterial isolates from China were compared with all known Xanthomonas type strains by a polyphasic approach including carbon source utilization profile, fatty acid methyl esters, analysis of 16S rRNA gene sequence and 16S–23S rDNA intergenic spacer sequences. The results indicated that these Chinese isolates could be further identified as a pathovar of Xanthomonas axonopodis.

Differentiation of Strains of Xylella fastidiosa Infecting Grape, Almonds, and Oleander Using a Multiprimer PCR Assay.

Xylella fastidiosa is a xylem-limited, nutritionally fastidious bacterium that causes several plant diseases including Pierce's disease (PD) in grape and leaf scorch in almond (ALS) and oleander (OLS). OLS strains belong to X. fastidiosa subsp. sandyi, PD strains belong to X. fastidiosa subsp. fastidiosa, and strains from almond designated as ALS strains are of two general types belonging either to X. fastidiosa subsp. multiplex or X. fastidiosa subsp. fastidiosa. The ALS strains assigned to X. fastidiosa subsp. multiplex belong to two different genotypes (ALSI and ALSII) below the subspecies level. The OLS strains do not infect grape or almond. PD strains produce diseases in grape, alfalfa, almond, and some weeds, but they do not infect oleander, oak, peach, or citrus. ALS strains that belong to X. fastidiosa subsp. multiplex do not produce disease on grape. In this study, a relatively simple polymerase chain reaction (PCR) based method was developed to distinguish among PD, OLS, and ALS strains. PCR performed with primers XF1968-L and XF1968-R amplified a 638-bp fragment from OLS strains but not from PD strains or ALS strains that belong to X. fastidiosa subsp. fastidiosa. PCR with primers XF2542-L and XF2542-R amplified a 412-bp fragment from PD strains, but not from OLS strains. PCR with primers ALM1 and ALM2 produced a fragment of 521 bp from strains isolated from almond that belong to X. fastidiosa subsp. multiplex. The combination of the three primer sets allowed the distinction of the two ALS genotypes of X. fastidiosa subsp. multiplex. These results are in agreement with those obtained from analysis of sequences of 16S-23S rDNA intergenic spacer regions sequence analysis and with previous results based on randomly amplified polymorphic DNA analysis.

Characterization of Lancefield group C Streptococcus dysgalactiae isolated from farmed fish

A Lancefield group C streptococcal (GCS) infection caused by Streptococcus dysgalactiae that is characterized by severe necrotic lesions of the caudal peduncle has been an increasing cause of mortality in farmed fish such as amberjack, Seriola dumerili, and yellowtail, Seriola quinqueradiata, in the southern part of Kyushu, Japan. In this study, enzymatic profiles of GCS strains from fish and mammals were investigated using the API ZYM system, and genotypic characterization of GCS strains was performed using biased sinusoidal field gel electrophoresis (BSFGE). The partial sequence of the 16S-23S rDNA intergenic spacer region of the GCS strain isolates from fish and mammals was also compared. The API ZYM test indicated that it is difficult to differentiate isolates of S. dysgalactiae from fish and animals based on enzymological variations. In the BSFGE analysis, the macrorestriction profiles, which were obtained using SmaI or ApaI as a restriction enzyme, revealed variations between the fish and animal isolates. The partial sequence of the 16S-23S rDNA intergenic spacer region of all the tested fish isolates differed from all mammalian isolates in one or two nucleotides. The possibility of a clonal expansion of S. dysgalactiae strains in farmed fish was also suggested by the BSFGE profiles of fish isolates.