Strain-specific Bands Research Articles

Phylogenetic analysis of heterocystous cyanobacteria (Subsections IV and V) using highly iterated palindromes as molecular markers.

Highly iterated palindromes (HIP) have been used as high resolution molecular markers for assessing the genetic variability and phylogenetic relatedness of heterocystous cyanobacteria (subsections IV and V) representing 12 genera of heterocystous cyanobacteria, collected from different geographical areas of India. DNA fingerprints generated using four HIP markers viz. HIP-AT, HIP-CA, HIP-GC, and HIP-TG showed 100% polymorphism in all the heterocystous cyanobacteria studied and each marker produced unique and strain-specific banding pattern. Furthermore, phylogenetic affinities based on the dendrogram constructed using HIP DNA profiles of heterocystous cyanobacteria suggest the monophyletic origin of this entire heterocystous clade along with a clear illustration of the polyphyletic origin of the branched Stigonematalean order (Subsection V). In addition, phylogenetic affinities were validated by principal component analysis of the HIP fingerprints. The overall data obtained by both the phylogeny and principal component assessments proved that the entire heterocystous clade was intermixed, and there are immediate needs for classificatory reforms that satisfy morphological plasticity and environmental concerns.

Morphological characterization and molecular fingerprinting of Nostoc strains by multiplex RAPD

Morphological parameters studied for the twenty selected Nostoc strains were mostly found to be consistent with the earlier reports. But the shape of akinetes observed in this study was a little deviation from the existing descriptions and heterocyst frequency was also found to be different in different strains in spite of growing in the same nitrogen free media. Multiplex RAPD produced reproducible and completely polymorphic amplification profiles for all the strains including some strain specific unique bands which are intended to be useful for identification of those strains. At least one to a maximum of two unique bands was produced by different dual primer combinations. For ten strains out of twenty, strain specific bands were found to be generated. Cluster analysis revealed a vast heterogeneity among these Nostoc strains and no specific clustering based on geographical origin was found except a few strains. It was also observed that morphological data may not necessarily correspond to the genetic data in most of the cases. CCC92 (Nostoc muscorum) and CCC48 (Nostoc punctiforme) showed a high degree of similarity which was well supported by high bootstrap value. The level of similarity of the strains ranged from 0.15 to 0.94. Cluster analysis based on multiplex RAPD showed a good fit revealing the discriminatory power of this technique.

A test for the “physiological phagemia” hypothesis—natural intestinal coliphages do not penetrate to the blood in horses

Soon after the discovery of bacteriophages in 1917, Felix d'Herelle demonstrated that they are present in humanand animal-associated microbiota, and speculated that these phages may play certain roles in the immunity against bacterial infections (d'Herelle 1926). That remains an intriguing but not yet proven possibility up to this day (reviewed in Gorski and Weber-Dabrowska 2005). It has been often reported that phages administrated orally or rectally in the course of phage therapy can penetrate through mucosa of gastrointestinal tract (GIT) to the blood (see Letarov et al. 2010; Gorski et al. 2006 for review) and it was suggested that natural intestinal bacteriophages might also translocate continuously from GIT to the blood. This state has been termed a “physiological phagemia” (Gorski et al. 2006). Coliphages, for example, were detected in a large fraction of commercial lots of animal sera used for eukaryotic cell cultures (reviewed in detail in Gorski et al. 2006). Since the bacteriophage communities present in different subjects are highly individual (Reyes et al. 2010; Golomidova et al. 2007; see also Letarov and Kulikov 2009), it is crucial to perform phage detection in the blood and in the intestinal contents simultaneously, using bacterial test strains able to detect the intestinal phages present in a particular subject to determine the extent to that intestinal phages in fact can routinely reach the blood. The aim of the work presented here is to quantify the presence of natural coliphages in the blood of the animals using a highly sensitive culturing approach. The choice of animal model was based on our data indicating that in contrast to humans and some other animal species (Reyes et al. 2010; see also refs in Letarov and Kulikov 2009), the intestinal viral community in horses is dominated by virulent phages that may occur in high titers in some healthy individuals (Golomidova et al. 2007; Kulikov et al. 2007; and our unpublished observations). Here, we present the analysis of bacteriophages in feces and in the blood of three horses. To select the animals for this study, we screened available population of horses for the titers of the fecal coliphages detectable on E.coli NM522 (Stratagene, USA) strain and selected three animals that yielded more than 10 plaque forming units (PFU)/g of coliphages. The indigenous coliform strains (ICSs) frequently detect different types of the coliphages in the same sample (Golomidova et al. 2007). In order to select a subset of these bacteria for subsequent phage detection, a series of 30 ICS isolates were obtained from each animal and subjected to whole-cell PCR fingerprinting using IS1tr (5′ATCAGTAAGTTGGA(G/A)(T/G)CATTACC-3′) and TR8834 (5′-ATCGGCGATGCGTTGACGAAT-3′) primers as described elsewhere (Isaeva et al. 2010; see also Golomidova et al. 2007). Briefly, the conventional PCR reactions (30 cycles consisting of 94°C for 15 s, 56°C for 30 s, 72°C for 45 s), using boiled cells from the plate as a template, were set up to generate strain-specific banding patterns. This analysis confirmed that all of the isolates were distinct, indicating M. Letarova and D. Strelkova contributed equally to this work. M. Letarova : S. Nevolina :A. Letarov (*) Winogradsky Institute of Microbiology RAS, Prospect 60-letiya Oktyabrya 7/2, 117312 Moscow, Russia e-mail: letarov@gmail.com

RAPD-SCAR Markers for Genetically Improved NEW GIFT Nile Tilapia (<I>Oreochromis niloticus niloticus</I> L.) and Their Application in Strain Identification

The NEW GIFT Nile tilapia (Oreochromis niloticus niloticus L.) is a nationally certificated new strain selected over 14 years and 9 generations from the base strain of GIFT Nile tilapia, introduced in 1994. This new variety has been extended in most of areas of China. The management of genetically improved strains, including the genetic markers for identification is needed urgently. RAPD analysis was conducted and their conversion to SCAR markers was developed. From NEW GIFT Nile tilapia, two strain-specific RAPD bands, S<SUB>304</SUB><SUP>624 bp</SUP> and S<SUB>36</SUB><SUP>568 bp</SUP> were identified. The strain-specific RAPD bands were gel-purified, cloned, and sequenced. Locus-specific primers were then designed to amplify the strain-specific bands. PCR amplification was conducted to test the variations in allele frequencies of two converted SCAR markers among the NEW GIFT Nile tilapia and its base strains, as well as 7 additional farmed strains worldwide. The frequency of SCAR marker I (553 bp) was 85.7% in NEW GIFT Nile tilapia, but 16.7% in the base strain. The frequency of SCAR marker II (558 bp) was 91.4% in NEW GIFT Nile tilapia, but 0% &#8722; 70% in the 7 other strains. In order to confirm the utility of these two markers, an examination was conducted for a wild population from Egypt, resulted the frequency of SCAR I and II was 10% and 70%, respectively, much lower than that of New GIFT strain. The increase in allele frequency of these two SCAR markers suggests that these markers might be genetically linked to the quantitative trait loci (QTL) underlining the performance traits by long term selection, and indicate the bright potential of SCAR marker technology for tracking generations during selection progress and for distinguishing among genetically improved strain and other strains.

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.

One stomach--one strain: does Helicobacter pylori strain variation influence disease outcome?

The aim of the study was to determine inter- and intrapatient variation of Helicobacter pylori strains based on genomic fingerprinting and cagA (cytotoxin-associated gene A) status. Ten bacterial colonies from each of 10 patients with gastric cancer (GC), 10 with duodenal ulcer (DU), and 10 with gastritis (GI) were used. The presence of the putative adhesin gene, the cagA gene, and the strain specific banding pattern obtained by arbitrary primed (AP-) PCR was analyzed. Genomic fingerprinting showed extensive interpatient variation, but the banding patterns obtained from colonies from the same patient were always identical (intrapatient variation). In five patients, the cagA status varied between the colonies despite identical banding patterns. Among patients in a developed country such as Sweden, the proportion with multiple-strain infection of H. pylori is low, but subclones with differing cagA status exist within the strain.

Genomic DNA fingerprinting by restriction fragment end labeling.

A typing method for bacteria was developed and applied to several species, including Escherichia coli and Actinobacillus actinomycetemcomitans. Total genomic DNA was digested with a restriction endonuclease, and fragments were enabled with [alpha-32P]dATP by using the Klenow fragment of DNA polymerase and separated by electrophoresis in 6% polyacrylamide/8 M urea (sequencing gel). Depending on the restriction endonuclease and the bacterium, the method produced approximately 30-50 well-separated fragments in the size range of 100-400 nucleotides. For A. actinomycetemcomitans, all strains had bands in common. Nevertheless, many polymorphisms could be observed, and the 31 strains tested could be classified into 29 distinct types. Furthermore, serotype-specific fragments could be assigned for the three serotypes investigated. The method described is very sensitive, allowing more distinct types to be distinguished than other commonly used typing methods. When the method was applied to 10 other clinically relevant bacterial species, both species-specific bands and strain-specific bands were found. Isolates from different locations of one patient showed indistinguishable patterns. Computer-assisted analysis of the DNA fingerprints allowed the determination of similarity coefficients. It is concluded that genomic fingerprinting by restriction fragment end labeling (RFEL) is a powerful and generally applicable technique to type bacterial species.

Invasion and intracellular development of theileria annulata sporozoites in lymphoblastoid cell lines already transformed by T. annulata (hissar), T. annulata (ankara) and T. parva (muguga)

Interactions between Theileria annulata sporozoites and lymphoblastoid cell lines already transformed by the Hissar and Ankara strains of T. annulata [T. a (H) and T.A. (A), respectively] and the Muguga strain of T. parva [T.P. (M)] were studied in vitro. Although sporozoites of the Hissar strain of T. annulata attached to and entered peripheral blood lymphocytes (PBL) and lymphoblastoid cell lines transformed by T. a (H) and T. a. (A), they neither attached to nor entered the T. p. (M) cell line. Whether the superinfecting T. a. (H) sporozoites developed intracellularly was studied by monitoring daily changes of mean schizont nuclear numbers and by determining electrophoretic mobilities of schizont glucose phosphate isomerase in each cell line using thin-layer starch gel electrophoresis. While the mean schizont nuclear number in freshly-infected PBL underwent a steady increase to the level of those in long standing T. annulata cultures, analysis of variance of similar data in T. a. (H) and T. a. (A) cell lines in which superinfection was demonstrated revealed no significant differences between them and their respective control counterparts, i.e., T. a. (H) and T. a. (A) cultures with no superinfection. Enzyme polymorphism studies showed the formation of uncontaminated species- or strain-specific bands of glucose phosphate isomerase (GPI) isoenzyme activity in the T. p. (M) and in the superinfected T. annulata cell lines.