Classification of 17 newly isolated virulent bacteriophages of Pseudomonas aeruginosa

Seventeen virulent bacteriophages specific to Pseudomonas aeruginosa strains were isolated by screening various environmental samples. These isolated bacteriophages were grouped based on results obtained from restriction fragment analysis of phage genomes, random amplification of polymorphic DNA (RAPD) typing, morphology observations under transmission electron microscope, and host range analysis. All 17 bacteriophages are double-stranded DNA viruses and can be divided into 5 groups based on DNA restriction profiles. A set of 10-mer primers was used in RAPD typing of phages, and similar conclusions were obtained as for restriction fragment analysis. One phage was randomly selected from each of the 5 groups for morphology observations. Four of them had an icosahedral head with a long contractile tail, belonging to the Myoviridae family, and one phage had an icosahedral head with a short tail, thereby belonging to the Podoviridae family. Host range experiments were conducted on 7 laboratory strains and 12 clinical strains of P. aeruginosa. The results showed that 13 phages had the same infection profile, killing 8 out of 19 tested P. aeruginosa strains, and the remaining 4 phages had different and unique infection profiles. This study highlights the diversity of bacteriophages specific to P. aeruginosa in the environment.

Differentiation of Paenibacillus larvae subsp. larvae, the Cause of American Foulbrood of Honeybees, by Using PCR and Restriction Fragment Analysis of Genes Encoding 16S rRNA

ABSTRACT A rapid procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that P. larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens formed a group with about 90% similarity; however, the P. larvae subsp. larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P. larvae subsp. larvae, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P. larvae subsp. larvae-infected larvae from all other species found in apiarian sources.