Lysogenicity of atypical strains ofSalmonella paratyphi B

Summary Atypical strains ofS. paratyphi B have been described which caused gastroenteritis or were found by chance in healthy perons. These strains possessed atypical natural phages. The late fermentation of d-tartrate by some strains ofS. paratyphi B was examined.     

HK022 bacteriophage Integrase mediated RMCE as a potential tool for human gene therapy

HK022 coliphage site-specific recombinase Integrase (Int) can catalyze integrative site-specific recombination and recombinase-mediated cassette exchange (RMCE) reactions in mammalian cell cultures. Owing to the promiscuity of the 7 bp overlap sequence in its att sites, active ‘attB’ sites flanking human deleterious mutations were previously identified that may serve as substrates for RMCE reactions for future potential gene therapy. However, the wild type Int proved inefficient in catalyzing such RMCE reactions. To address this low efficiency, variants of Int were constructed and examined by integrative site-specific recombination and RMCE assays in human cells using native ‘attB’ sites. As a proof of concept, various Int derivatives have demonstrated successful RMCE reactions using a pair of native ‘attB’ sites that were inserted as a substrate into the human genome. Moreover, successful RMCE reactions were demonstrated in native locations of the human CTNS and DMD genes whose mutations are responsible for Cystinosis and Duchene Muscular Dystrophy diseases, respectively. This work provides a steppingstone for potential downstream therapeutic applications.     

Multi-locus sequence typing: a tool for global epidemiology

The characterization of pathogenic isolates plays a pivotal role in the epidemiology of infectious diseases, generating the information necessary for identifying, tracking, and intervening against disease outbreaks. In 1998 multi-locus sequence typing (MLST) was proposed as a nucleotide sequence-based approach that could be applied to many bacterial pathogens. It combined developments in high-throughput sequencing and bioinformatics with established population genetics techniques to provide a portable, reproducible, and scalable typing system that reflected the population and evolutionary biology of bacterial pathogens. MLST schemes have been developed for a variety of procaryotic and eucaryotic pathogens and the data generated have contributed to both epidemiological surveillance and fundamental studies of pathogen biology.     

PHYPred:a tool for identifying bacteriophage enzymes and hydrolases

Dear Editor,Bacteriophages are viruses that attack bacteria and kill them through the lytic replication cycle.Many studies have reported that phages are more specific to bacteria than antibiotics are;thus,phage therapy has many potential applications in human medicine,with the advantage of having few side effects(Keen,2012).Investigating the mechanisms of bacteria-killing phages will therefore aid     

Genomic methylation: a tool for typing Helicobacter pylori isolates

The genome sequences of three Helicobacter pylori strains revealed an abundant number of putative restriction and modification (R-M) systems within a small genome (1.60 to 1.67 Mb). Each R-M system includes an endonuclease that cleaves a specific DNA sequence and a DNA methyltransferase that methylates either adenosine or cytosine within the same DNA sequence. These are believed to be a defense mechanism, protecting bacteria from foreign DNA. They have been classified as selfish genetic elements; in some instances it has been shown that they are not easily lost from their host cell. Possibly because of this phenomenon, the H. pylori genome is very rich in R-M systems, with considerable variation in potential recognition sequences. For this reason the protective aspect of the methyltransferase gene has been proposed as a tool for typing H. pylori isolates. We studied the expression of H. pylori methyltransferases by digesting the genomic DNAs of 50 strains with 31 restriction endonucleases. We conclude that methyltransferase diversity is sufficiently high to enable the use of the genomic methylation status as a typing tool. The stability of methyltransferase expression was assessed by comparing the methylation status of genomic DNAs from strains that were isolated either from the same patient at different times or from different stomach locations (antrum and corpus). We found a group of five methyltransferases common to all tested strains. These five may be characteristic of the genetic pool analyzed, and their biological role may be important in the host/bacterium interaction.     

Faecal DNA typing as a tool for investigating territorial behaviour of badgers (Meles meles)

We use data from three social groups of badgers (Meles meles) to illustrate how faecal DNA genotyping could be used in scent-marking studies. Faecal samples collected from latrines were genotyped to determine the individual identity and sex of badgers engaging in territorial behaviour and the frequency with which those individuals defecated at particular latrines. The method is potentially applicable to other species of carnivores that use latrines to mark their territories.     

Resistogram typing as an epidemiological tool for Escherichia coli isolates of poultry origin

A rapid method for the detection of corynetoxins, tunicamycin-like antibiotics, is described. Test samples were applied to or grown on an agar medium and overlain with Clavibacter tritici which is highly sensitive to the toxins. The method could detect 50 ng of tunicamycin. Corynetoxins in a range of field and laboratory samples were readily detected.     

Biochemical typing as a method of differentiating group B streptococcus]

In epidemiological studies on the group B streptococcus the serological typing is used. The paper present the results of a study on usefulness of biochemical typing for differentiation of the group B streptococcus. For that purpose, 210 strains descended from colonized infants and pregnant women were put to typing with both of mentioned methods. We showed that each of the method distinguishes similar number of biotypes and serotypes. However, ought to be marked that significant number of strains (93.8%) belonged to the three out of eight biochemical types. Similar results were achieved in serological typing, three of the most numerous serotypes contained 81.4% strains. Analysis of the relationship between serological and biochemical types did not reveal statistical association because the strains belonged to various serotypes. Obtained results show that both methods of typing--biochemical and serological, have similar value in differentiation of the strains. The method of biochemical typing is quite simple and can be used in laboratory conditions.     

Restriction endonuclease BglI as a tool for in vitro reconstruction and recombination of plasmid and bacteriophage genomes

The restriction endonuclease BglI produces different individual fragment ends from different cut sites. This property has allowed us to reconstruct efficiently several commonly used plasmid and bacteriophage genomes and a number of recombinant plasmids containing up to seven BglI restriction sites from their constituent BglI fragments. It is demonstrated that in vitro reconstitution from BglI fragments can be used to create, in a simple way, recombinant DNA molecules by recombining in vitro BglI fragments from different mutated or otherwise related genomes. Further applications of the method are discussed.     

Subtracted restriction fingerprinting--a tool for bacterial genome typing

Reproducible, discriminative, high-throughput methods are required for the identification of bacterial strains and isolates in a clinical environment. A new molecular typing method for bacteria was developed and tested on Salmonella and E. coli species. The technique is called subtracted restriction fingerprinting and is based on double restriction enzyme digestion of genomic DNA followed by end labeling. The "detection" enzyme produces TTAA overhangs that are filled in with digoxigenated nucleotides for subsequent detection, while the "subtraction" enzyme produces GCGC overhangs that are filled in with biotinylated nucleotides that permit the removal of this subset of fragments with either streptavidin-coated magnetic particles or AffiniTip streptavidin columns. The two restriction enzymes are selected to produce a fragment size profile suitable for a specific analytical system. In this demonstration of the principle of subtracted restriction fingerprinting, analysis of Salmonella enterica subsp. enterica serovar Dublin and E. coli on a 30-cm 1.2% agarose gel revealed up to 50 sharp evenly spaced bands, which were sufficient for the discrimination between various isolates and substrains. The restriction enzyme combinations suitable for the analysis of Salmonella and E. coli are presented. The method requires fewer enzymatic steps than amplified fragment length polymorphism, does not need the specialized DNA preparation essential for pulsed field gel electrophoresis, and has a higher reproducibility than PCR-based methods.     

Development of a multilocus sequence typing tool for high-resolution genotyping of Enterocytozoon bieneusi

Thus far, genotyping of Enterocytozoon bieneusi has been based solely on DNA sequence analysis of the internal transcribed spacer (ITS) of the rRNA gene. Both host-adapted and zoonotic (human-pathogenic) genotypes of E. bieneusi have been identified. In this study, we searched for microsatellite and minisatellite sequences in the whole-genome sequence database of E. bieneusi isolate H348. Seven potential targets (MS1 to MS7) were identified. Testing of the seven targets by PCR using two human-pathogenic E. bieneusi genotypes (A and Peru10) led to the selection of four targets (MS1, MS3, MS4, and MS7). Further analysis of the four loci with an additional 24 specimens of both host-adapted and zoonotic E. bieneusi genotypes indicated that most host-adapted genotypes were not amplified by PCR targeting these loci. In contrast, 10 or 11 of the 13 specimens of the zoonotic genotypes were amplified by PCR at each locus. Altogether, 12, 8, 7, and 11 genotypes of were identified at MS1, MS3, MS4, and MS7, respectively. Phylogenetic analysis of the nucleotide sequences obtained produced a genetic relationship that was similar to the one at the ITS locus, with the formation of a large group of zoonotic genotypes that included most E. bieneusi genotypes in humans. Thus, a multilocus sequence typing tool was developed for high-resolution genotyping of E. bieneusi. Data obtained in the study should also have implications for understanding the taxonomy of Enterocytozoon spp., the public health significance of E. bieneusi in animals, and the sources of human E. bieneusi infections.     

Siderophore typing,a powerful tool for the identification of fluorescent and nonfluorescent pseudomonads

A total of 301 strains of fluorescent pseudomonads previously characterized by conventional phenotypic and/or genomic taxonomic methods were analyzed through siderotyping, i.e., by the isoelectrophoretic characterization of their main siderophores and pyoverdines and determination of the pyoverdine-mediated iron uptake specificity of the strains. As a general rule, strains within a well-circumscribed taxonomic group, namely the species Pseudomonas brassicacearum, Pseudomonas fuscovaginae, Pseudomonas jessenii, Pseudomonas mandelii, Pseudomonas monteilii, "Pseudomonas mosselii," "Pseudomonas palleronii," Pseudomonas rhodesiae, "Pseudomonas salomonii," Pseudomonas syringae, Pseudomonas thivervalensis, Pseudomonas tolaasii, and Pseudomonas veronii and the genomospecies FP1, FP2, and FP3 produced an identical pyoverdine which, in addition, was characteristic of the group, since it was structurally different from the pyoverdines produced by the other groups. In contrast, 28 strains belonging to the notoriously heterogeneous Pseudomonas fluorescens species were characterized by great heterogeneity at the pyoverdine level. The study of 23 partially characterized phenotypic clusters demonstrated that siderotyping is very useful in suggesting correlations between clusters and well-defined species and in detecting misclassified individual strains, as verified by DNA-DNA hybridization. The usefulness of siderotyping as a determinative tool was extended to the nonfluorescent species Pseudomonas corrugata, Pseudomonas frederiksbergensis, Pseudomonas graminis, and Pseudomonas plecoglossicida, which were seen to have an identical species-specific siderophore system and thus were easily differentiated from one another. Thus, the fast, accurate, and easy-to-perform siderotyping method compares favorably with the usual phenotypic and genomic methods presently necessary for accurate identification of pseudomonads at the species level.     

Nuclease-coated bacteriophage: a sensitive tool for studying antigenic reactivity of synthetic sequence fragments

Staphylococcal nuclease was conjugated to bacteriophage T₄ using glutaraldehyde as a cross-linking agent. The conjugated phage is inactivated by antinuclease antibodies and this inactivation is specifically inhibited by nuclease in concentrations as low as 10^?9m. Two fragments of the enzyme, namely P₂ (residues 6–48) and P₃ (residues 49–149) inhibit to a much lower extent the inactivation of the conjugated bacteriophage by the antibodies, than the native enzyme, but when mixed together (forming the noncovalent complex, nuclease-T), the inhibition curve obtained is similar to that obtained by native nuclease. This sensitive system was applied for testing different synthetic sequence fragments and for studying the complementarity of synthetic sequences in the P₂ region with native P₃.