Restriction site tagged (RST) microarrays: a novel technique to study the species composition of complex microbial systems.

We have developed a new type of microarray, restriction site tagged (RST), for example NotI, microarrays. In this approach only sequences surrounding specific restriction sites (i.e. NotI linking clones) were used for generating microarrays. DNA was labeled using a new procedure, NotI representation, where only sequences surrounding NotI sites were labeled. Due to these modifications, the sensitivity of RST microarrays increases several hundred-fold compared to that of ordinary genomic microarrays. In a pilot experiment we have produced NotI microarrays from Gram-positive and Gram-negative bacteria and have shown that even closely related Escherichia coli strains can be easily discriminated using this technique. For example, two E.coli strains, K12 and R2, differ by less than 0.1% in their 16S rRNA sequences and thus the 16S rRNA sequence would not easily discriminate between these strains. However, these strains showed distinctly different hybridization patterns with NotI microarrays. The same technique can be adapted to other restriction enzymes as well. This type of microarray opens the possibility not only for studies of the normal flora of the gut but also for any problem where quantitative and qualitative analysis of microbial (or large viral) genomes is needed.

DNA-based classification and sequence heterogeneities in the 16S rRNA genes of Lactobacillus casei/paracasei and related species.

The sequence differences within the 16S rRNA genes of Lactobacillus casei/paracasei and related species, Lactobacillus zeae and Lactobacillus rhamnosus, were investigated. Thirty-seven strains of mostly human or cheese origin were grouped by restriction endonuclease analysis (REA) of the total chromosomal DNA and by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rRNA gene fragments. REA verified that all strains were genomically unique and singled out three major clusters, one L. rhamnosus-cluster and two clusters containing L. paracasei strains. The groups obtained by TTGE corresponded with one exception to the REA-clusters. In the TTGE clustering all L. paracasei strains formed one general group with one TTGE-band in common, and this group was sub-divided into five subgroups due to the presence of more than one TTGE-band in four of the subgroups. The occurrence of multiple TTGE-bands was investigated by amplifying and cloning of the 16S rRNA genes from the strains showing this phenomenon, thereby 12 clones from each strain were sequenced, demonstrating polymorphisms in almost all the cases. Subjecting the clones displaying sequence variations to TTGE as well as sequencing of 16S rDNA revealed by ribotyping of the strains, verified the presence of polymorphisms within the 16S rRNA genes. The migration characteristic of amplified DNA from a single clone corresponded to a specific band in the TTGE-pattern of the strain from which the clone originated. Southern blot hybridisation with a 16S rDNA probe demonstrated the presence of at least five 16S rRNA genes in L. casei/paracasei. A higher degree of variable positions than previously reported was observed in the 16S rRNA gene fragments of the members in the complex. Sequence comparison between the 16S rRNA gene copies of L. casei (CCUG 21451T) and L. zeae (CCUG 35515T) demonstrated that the two species shared almost the same sequence in some copies while the others were more different. Our results provide one explanation for the difficulties in reaching clear-cut taxa within the L. casei/paracasei complex.

ISMmy1, a novel insertion sequence of Mycoplasma mycoides subsp. mycoides small colony type.

A new insertion sequence, ISMmy1, has been identified in the bovine pathogen Mycoplasma mycoides subsp. mycoides biotype small colony (MmymySC). The occurrence of ISMmy1 in 15 MmymySC strains and 12 other mycoplasmas was examined by Southern blotting. All MmymySC strains showed identical hybridisation patterns except for the type strain PG1(T), the vaccine strain T1Sr49, and the strain Afadé, which all had unique patterns. ISMmy1-like sequences were also found in the bovine pathogen Mycoplasma bovis strain Donetta(T) while mycoplasmas that are phylogenetically closer to MmymySC lack ISMmy1. This observation suggests horizontal transfer between MmymySC and M. bovis.

Brachyspira hyodysenteriae and other strongly beta-haemolytic and indole-positive spirochaetes isolated from mallards (Anas platyrhynchos).

The aims of the current study were to collect intestinal spirochaetes (genus Brachyspira) from farmed and wild mallards (Anas platyrhynchos) and to identify and classify those isolates that phenotypically resembled Brachyspira hyodysenteriae, an enteric pathogen of pigs. The isolation rate of Brachyspira spp. was high from both farmed (93 %) and wild mallards (78 %). In wild mallards, it appeared that Brachyspira spp. were more likely to be found in migratory birds (multivariate analysis: RR = 1.8, 95 % CI 1.1-3.1) than in mallards sampled in a public park. Pure cultures of putative B. hyodysenteriae were obtained from 22 birds. All five isolates from farmed mallards and ten randomly selected isolates with this phenotype were used for further studies. All isolates from farmed mallards and two of the isolates from wild mallards were PCR-positive for the tlyA gene of B. hyodysenteriae. Two isolates from farmed mallards were selected for pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis. These isolates clustered with the type and reference strains of B. hyodysenteriae. 16S rDNA sequence analysis performed on 11 of the strains showed that they were all closely related to each other and to the B. hyodysenteriae-Brachyspira intermedia cluster. Three of the mallard isolates had 16S rDNA sequences that were identical to those of B. hyodysenteriae strains R1 and NIV-1 previously isolated from common rheas (Rhea americana). To conclude, the isolates from farmed mallards and two isolates from wild mallards were classified as B. hyodysenteriae based on the fact that they could not be differentiated by any of the applied methods from type, reference and field strains of B. hyodysenteriae. The remaining isolates could not be assigned irrefutably to any of the presently recognized Brachyspira species. These results point to a broader host spectrum of B. hyodysenteriae than is generally recognized, and to the presence in mallards of strongly beta-haemolytic and indole-producing spirochaetes that possess many, but not all, of the currently recognized characteristics of B. hyodysenteriae.

Identification of three clusters of canine intestinal spirochaetes by biochemical and 16S rDNA sequence analysis.

It has been suggested that canine intestinal spirochaetes consist of Brachyspira pilosicoli and a group of strains that has been provisionally designated 'Brachyspira canis'. The purpose of the present study was to compare 22 spirochaete isolates that were obtained from intestinal specimens of dogs in Sweden (n = 12), Norway (n = 4), the United States (n = 3), Australia (n = 2) and Germany (n = 1) with type and reference strains, as well as field isolates, of Brachyspira species by five biochemical tests and determination of almost-complete 16S rDNA sequences. In an evolutionary tree derived from 16S rDNA sequences, the canine isolates grouped into three clusters. One cluster included the type strain of porcine B. pilosicoli, whereas a second larger cluster, which was monophyletic, contained a canine strain that was identified previously as 'B. canis'. The third cluster consisted of three canine isolates of Scandinavian origin, which grouped together with the type strain of the species Brachyspira alvinipulli (pathogenic to chicken). These three genotypes, which were identified on the basis of 16S rDNA sequences, corresponded to four phenotypic groups based on biochemical testing. Two biochemical tests, hippurate hydrolysis and alpha-galactosidase production, were sufficient for rapid identification of each canine cluster.

DNA based classification of food associated Enterobacteriaceae previously identified by Biolog GN Microplates.

Enterobacteriaceae are frequently isolated from food products and it is essential to have methods for correct identification for both food hygiene and epidemiology reasons. Phenotypic methods are not always sufficient and have to be supplemented by DNA based methods. In the present study, 70 strains of Enterobacteriaceae derived from milk, fish and meat that had previously been identified by Biolog GN Microplates were genomically classified together with 15 representative type strains of species of Enterobacteriaceae. The field strains were dominated by Hafnia alvei, Serratia liquefaciens and Rahnella aquatilis. All strains were subjected to temporal temperature gel electrophoresis (TTGE) analysis using amplicons encompassing the V3, V4 and V9 variable regions of the 16S rRNA gene. Selected strains were analysed by ribotyping and partial 16S rDNA sequencing. The type strains were differentiated into 10 different TTGE groups. Two of the groups contained two type strains. Enterobacter aerogenes and Klebsiella planticola were not distinguished due to their identical sequences and Yersinia ruckeri and Citrobacter freundii showed the same migration pattern. The 70 food strains could be differentiated into 14 TTGE groups where 33 strains (47.1%) could be assigned to TTGE groups including type or reference strains. Rahnella strains were dispersed into three TTGE groups of which one group corresponded to Rahnella genomospecies 1 and one to genomospecies 3. The grouping of Rahnella strains was supported by ribotyping and phylogenetic analysis. TTGE can be a useful additional tool for identification on the species level of food related Enterobacteriaceae.

The bacterial flora of fresh and chill-stored pork: analysis by cloning and sequencing of 16S rRNA genes.

The composition of the initial bacterial flora of pork and the development of the flora after storage at +4 degrees C for 4 days were analysed by amplification, cloning and sequencing of 16S rDNA. A total of 122 clones were obtained, with lengths of > or =400 nucleotides and > or =95% similarity to database sequences. Nineteen clones were similar to sequences in database not assigned to any genera. Fourteen different genera were represented in clones from fresh meat, with 36.5% of the clones most resembling Acinetobacter and 17.3% resembling Staphylococcus and Macrococcus. After storage, the clones were composed of six different genera, with 44.3% resembling Pseudomonas, 17.1% resembling Aeromonas and only 14.3% resembling Acinetobacter. This study shows that the overall pattern of the initial and chill-stored pork flora, as shown by a molecular approach, was in agreement with results obtained in previous studies using traditional cultivation methods.

Characterization of lactobacilli from Scotch malt whisky distilleries and description of Lactobacillus ferintoshensis sp. nov., a new species isolated from malt whisky fermentations.

Sixty-four strains of Lactobacillus were isolated from fermentation samples from 23 malt whisky distilleries located in the major whisky producing regions of Scotland. The strains were assigned to 26 ribotype patterns. Strains of some ribotype patterns were widely distributed and recovered from distilleries throughout Scotland, while strains representing other ribotypes were particular to a specific region or even a certain distillery. Repeated sampling of a single distillery over a 12 month period showed that the range of bacteria present, as indicated by ribotyping, was stable, but was influenced by changes in malt supply and the period of closure for annual maintenance. Partial 16S rDNA sequence analysis of ribotype representatives revealed Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus paracasei and Lactobacillus pentosus as the major species present in the distilleries; however, four isolates could not be identified by this procedure. Determination of the full 16S rDNA gene sequence from one of these isolates (strain R7-84) revealed >98.5% similarity to Lactobacillus buchneri and its phylogenetic neighbours. DNA from two other strains showed greater than 70% hybridization to DNA from R7-84 under non-stringent renaturation conditions and DNA from strain R7-84 shared less than 65% hybridization with members of the L. buchneri group. It is proposed that these three strains should be placed in a new species for which the name Lactobacillus ferintoshensis represented by the type strain R7-84(T) is suggested.

Bacillus endophyticus sp. nov., isolated from the inner tissues of cotton plants (Gossypium sp.).

Four strains of aerobic, endospore-forming bacteria were isolated from the inner tissues of healthy cotton plants (Gossypium sp., Dushanbe, Tajikistan). The organisms had identical randomly amplified polymorphic DNA patterns that distinguished them from other bacilli that are commonly isolated from plant tissues, e.g. Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus and Bacillus subtilis. PCR amplification of 16S-23S rRNA spacer regions suggested that the four strains could be assigned to two highly related taxa, which correlated with differences in cell morphology. However, the cloned spacer region provided a simple and specific hybridization probe for all four strains. The virtually complete 16S rDNA sequences were prepared for representatives of the two groups (strains 2DT(T) and 12DX) and differed by only two bases, thus supporting classification of the four strains in a single taxon at the species level. Phylogenetic analyses indicated that strain 2DT(T) belonged to the genus Bacillus and was most closely related to Bacillus sporothermodurans DSM 10599T with a sequence similarity of 94.8%. It is concluded that the four strains belong to a novel species of Bacillus for which the name Bacillus endophyticus sp. nov. is proposed. The type strain is 2DT(T) (= UCM B-5715T = CIP 106778T).

The genome sequence of Mycoplasma mycoides subsp. mycoides SC type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP).

Mycoplasma mycoides subsp. mycoidesSC (MmymySC)is the etiological agent of contagious bovine pleuropneumonia (CBPP), a highly contagious respiratory disease in cattle. The genome of Mmymy SC type strain PG1(T) has been sequenced to map all the genes and to facilitate further studies regarding the cell function of the organism and CBPP. The genome is characterized by a single circular chromosome of 1211703 bp with the lowest G+C content (24 mole%)and the highest density of insertion sequences (13% of the genome size)of all sequenced bacterial genomes. The genome contains 985 putative genes, of which 72 are part of insertion sequences and encode transposases. Anomalies in the GC-skew pattern and the presence of large repetitive sequences indicate a high genomic plasticity. A variety of potential virulence factors was identified, including genes encoding putative variable surface proteins and enzymes and transport proteins responsible for the production of hydrogen peroxide and the capsule, which is believed to have toxic effects on the animal.

Whole cell protein and partial 16S rRNA gene sequence analysis suggest the existence of a second Rothia species.

OBJECTIVE: To subject ten clinical isolates grouped together based on their biochemical and microbiological profile to further investigations aimed at correct species identification. METHODS: The 16S rRNA gene was partially sequenced using nested amplification. Whole cell protein analysis (SDS-PAGE) and cluster analysis were performed on the 10 strains and also for comparison on 31 reference strains. The API Coryne biochemical kit as well as API 20 Strep were used for analysis of the phenotypic diversity of the strains by use of computerized numerical identification procedures. Antibiotic susceptibility testing was performed using a standardized disk diffusion test. RESULTS: The 265--556-bp-long 16S rRNA gene sequences of all 10 strains showed highest similarity to Rothia dentocariosa. Three strains showed complete identity between the sequences obtained and the sequence of the type strain of Rothia dentocariosa 16S rRNA gene (M59055), and the other seven ranged between 99.7% and 98.3% similarity. Detailed analysis of the sequences revealed a clustering of the strains into two groups. One group consisted of four isolates with the highest degrees of similarity with the reference strain (type I), while the members of another group (type II) showed differences in their nucleotide sequence at four distinct positions in the variable V7 region. T was replaced by C at position 597, C by T at position 608, T by C at position 610, and G by A at position 684 (position numbers according to reference sequence M59055, EMBL/GenBank). Whole cell protein analysis (SDS-PAGE) and cluster analysis also segregated the 10 Rothia dentocariosa strains into two different clusters, with one cluster containing all four strains belonging to 16S rRNA gene type I, and a second cluster containing all six strains belonging to 16S rRNA gene type II. CONCLUSIONS: Partial sequence data of the 16S rRNA gene as well as whole cell protein analysis showed a subdivision of the Rothia species into two groups, genomovar I (Rothia dentocariosa sensu stricto) and genomovar II, a possible new Rothia species.