Intragenus generalized transduction in Staphylococcus spp. by a novel giant phage.

Bacteriophage (phage)-mediated generalized transduction is expected to contribute to the emergence of drug-resistant staphylococcal clones in various environments. In this study, novel phage S6 was isolated from sewage and used to test generalized transduction in human- and animal-derived staphylococci. Phage S6 was a novel type of giant myophage, which possessed a DNA genome that contained uracil instead of thymine, and it could infect all of the tested staphylococcal species. The phage S6 appeared to be similar to the transducing phage PBS1, which infects Bacillus spp. Moreover, phage S6 facilitated the transduction of a plasmid in Staphylococcus aureus and from S. aureus to non-aureus staphylococcal species, as well as vice versa. Transduction of methicillin resistance also occurred in S. aureus. This is the first report of successful intragenus generalized transduction among staphylococci.

Characterization of Helicobacter pylori bacteriophage KHP30.

Helicobacter pylori inhabits the stomach mucosa and is a causative agent of stomach ulcer and cancer. In general, bacteriophages (phages) are strongly associated with bacterial evolution, including the development of pathogenicity. Several tailed phages have so far been reported in H. pylori. We have isolated an H. pylori phage, KHP30, and reported its genomic sequence. In this study, we examined the biological characteristics of phage KHP30. Phage KHP30 was found to be a spherical lipid-containing phage with a diameter of ca. 69 nm. Interestingly, it was stable from pH 2.5 to pH 10, suggesting that it is adapted to the highly acidic environment of the human stomach. Phage KHP30 multiplied on 63.6% of clinical H. pylori isolates. The latent period was ca. 140 min, shorter than the doubling time of H. pylori (ca. 180 min). The burst size was ca. 13, which was smaller than the burst sizes of other known tailed or spherical phages. Phage KHP30 seemed to be maintained as an episome in H. pylori strain NY43 cells, despite a predicted integrase gene in the KHP30 genomic sequence. Seven possible virion proteins of phage KHP30 were analyzed using N-terminal protein sequencing and mass spectrometry, and their genes were found to be located on its genomic DNA. The genomic organization of phage KHP30 differed from the genomic organizations in the known spherical phage families Corticoviridae and Tectiviridae. This evidence suggests that phage KHP30 is a new type of spherical phage that cannot be classified in any existing virus category.

Analysis of deoxynucleosides in bacteriophages ϕEF24C and K and the frequency of a specific restriction site in the genomes of members of the bacteriophage subfamily Spounavirinae.

The genera SPO1-like and Twort-like viruses in the subfamily Spounavirinae of the family Myoviridae have been newly proposed, with the reorganization of the SPO1-related bacteriophages (phages). A criterion defining these viral genera is the presence/absence of DNA modifications. In this study, liquid chromatography/mass spectrometry showed that phages ϕEF24C and K of the subfamily Spounavirinae have unmodified DNA, which classifies them as Twort-like viruses. Moreover, in the subfamily Spounavirinae, DNA modification and elimination of a particular DNA sequence were suggested to be the major antirestriction strategies of the SPO1-like and Twort-like viruses, respectively.

Improved liquid chromatography/mass spectrometric analysis of low molecular weight carboxylic acids by ion exclusion separation with electrospray ionization.

An improved, simple and sensitive analytical method for low molecular weight organic acids has been developed. A mixture of acetic, propionic, butyric, glycolic, lactic, 2-hydroxybutyric, malonic, succinic, glutaric, tartaric and citric acids was separated on a semi-rigid styrene-divinylbenzene copolymer-based H-type cation-exchange resin (ULTRON PS-80H) based on an ion exclusion chromatographic (IEC) mechanism, with detection using electrospray ionization mass spectrometry (ESI-MS). Formic or acetic acid was used as a mobile phase to separate the carboxylic acids within 15 min. For liquid chromatography/mass spectrometry (LC/MS), the ESI interface was used in both positive and negative ionization mode. ESI produced reasonable signals from positive ions, [M+NH(4)](+), of acetic, propionic and butyric acids and from negative ions, [M-H](-), of glycolic, lactic, 2-hydroxybutyric, malonic, succinic, glutaric, tartaric and citric acids. The effects of ionization parameters, source temperature, capillary voltage and cone voltage, on sensitivity and linearity were examined. Linear plots of peak area versus concentration were obtained over the range 0.1-20 ppm for MS detection. The detection limits of the target carboxylic acids calculated at signal-to-noise (S/N) ratio of 3 ranged from 9 to 59 ppb. The reproducibility of retention times and peak areas were 0.55-1.25 and 0.85-2.45%, respectively.

Effect of alcohol addition on the movement of petroleum hydrocarbon fuels in soil.

Groundwater contamination by fuel spills from aboveground and underground storage tanks has been of growing concern in recent years. This problem has been magnified by the addition of oxygenates, such as ethanol and methyl-tertiary-butyl ether (MTBE) to fuels to reduce vehicular emissions to the atmosphere. These additives, although beneficial in reducing atmospheric pollution, may, however, increase groundwater contamination due to the co-solvency of petroleum hydrocarbons and by the provision of a preferential substrate for microbial utilisation. With the introduction of ethanol to diesel fuel imminent and the move away from MTBE use in many states of the USA, the environmental implications associated with ethanol additive fuels must be thoroughly investigated. Diesel fuel movement was followed in a 1-m soil column and the effect of ethanol addition to diesel fuel on this movement determined. The addition of 5% ethanol to diesel fuel was found to enhance the downward migration of the diesel fuel components, thus increasing the risk of groundwater contamination. A novel method using soil packed HPLC columns allowed the influence of ethanol on individual aromatic hydrocarbon movement to be studied. The levels of ethanol addition investigated were at the current additive level (approx. 25%) for ethanol additive fuels in Brazil and values above (50%) and below (10%) this level. An aqueous ethanol concentration above 10% was required for any movement to occur. At 25% aqueous ethanol, the majority of hydrocarbons were mobilised and the retention behaviour of the soil column lessened. At 50% aqueous ethanol, all the hydrocarbons were found to move unimpeded through the columns. The retention behaviour of the soil was found to change significantly when both organic matter content and silt/clay content was reduced. Unexpectedly, sandy soil with low organic matter and low silt/clay was found to have a retentive behaviour similar to sandy subsoil with moderate silt/clay, but little organic matter. It was concluded that sand grains might have a more important role in the adsorption of petroleum hydrocarbons than first realised. This method has shown that soil packed HPLC columns can be used to provide a quick estimate of petroleum hydrocarbon, and possibly other organic contaminant, movement in a variety of different soil types.