rbcL sequences reveal multiple cryptic introductions of the Japanese red alga Polysiphonia harveyi.

In Europe, the last 20 years have seen a spectacular increase in accidental introductions of marine species, but it has recently been suggested that both the actual number of invaders and their impacts have been seriously underestimated because of the prevalence of sibling species in marine habitats. The red alga Polysiphoniaharveyi is regarded as an alien in the British Isles and Atlantic Europe, having appeared in various locations there during the past 170 years. Similar or conspecific populations are known from Atlantic North America and Japan. To choose between three competing hypotheses concerning the origin of P. harveyi in Europe, we employed rbcL sequence analysis in conjunction with karyological and interbreeding data for samples and isolates of P. harveyi and various congeners from the Pacific and North Atlantic Oceans. All cultured isolates of P. harveyi were completely interfertile, and there was no evidence of polyploidy or aneuploidy. Thus, this biological species is both morphologically and genetically variable: intraspecific rbcL divergences of up to 2.1% are high even for red algae. Seven rbcL haplotypes were identified. The four most divergent haplotypes were observed in Japanese samples from Hokkaido and south-central Honshu, which are linked by hypothetical 'missing' haplotypes that may be located in northern Honshu. These data are consistent with Japan being the centre of diversity and origin for P. harveyi. Two non-Japanese lineages were linked to Hokkaido and Honshu, respectively. A single haplotype was found in all North Atlantic and Mediterranean accessions, except for North Carolina, where the haplotype found was the same as that invading in New Zealand and California. The introduction of P. harveyi into New Zealand has gone unnoticed because P. strictissima is a morphologically indistinguishable native sibling species. The sequence divergence between them is 4–5%, greater than between some morphologically distinct red algal species. Two different types of cryptic invasions of P. harveyi have therefore occurred. In addition to its introduction as a cryptic sibling species in New Zealand, P. harveyi has been introduced at least twice into the North Atlantic from presumed different source populations. These two introductions are genetically and probably also physiologically divergent but completely interfertile.

The inactivation of bovine cathepsin B by novel N-chloro-acetyl-dipeptides. Application of the Houghten 'tea bag' methodology to inhibitor synthesis

In this study, a series of N-chloro-acetylated dipeptides were synthesised by the application of Houghten's methodology of multiple analog peptide syntheses. The peptides, all of which contain a C-terminal free acid, were tested as inactivators of bovine cathepsin B, in an attempt at exploiting the known and, amongst the cysteine proteinases, unique carboxy dipeptidyl peptidase activity of the protease. We have succeeded in obtaining a number of effective inactivators, the most potent of which-chloroacetyl-Leu-Leu-OH, inactivates the enzyme with an apparent second-order rate constant of 3.8 x 10(4) M-1 min(-1). In contrast, the esterified analog, chloroacetyl-Leu-Leu-OMe, inactivates the enzyme some three orders of magnitude less efficiently, lending credence to our thesis that a free carboxylic acid moiety is an important determinant for inhibitor effectiveness. This preliminary study has highlighted a number of interesting features about the specificity requirements of the bovine proteinase and we believe that our approach has great potential for the rapid delineation of the subsite specificities of cathepsin B-like proteases from various species. (c) 2005 Elsevier Inc. All rights reserved.

In vitro activity of tea-tree oil against clinical skin isolates of meticillin-resistant and -sensitive Staphylococcus aureus and coagulase-negative staphylococci growing planktonically and as biofilms

The susceptibility of Staphylococcus aureus [meticillin-resistant (MRSA) and meticillin-sensitive (MSSA)] and coagulase-negative staphylococci (CoNS), which respectively form part of the transient and commensal skin flora, to tea-tree oil (TTO) was compared using broth microdilution and quantitative in vitro time-kill test methods. MRSA and MSSA isolates were significantly less susceptible than CoNS isolates, as measured by both MIC and minimum bactericidal concentration. A significant decrease in the mean viable count of all isolates in comparison with the control was seen at each time interval in time-kill assays. However, the only significant difference in the overall mean log(10) reduction in viable count between the groups of isolates was between CoNS and MSSA at 3 h, with CoNS isolates demonstrating a significantly lower mean reduction. To provide a better simulation of in vivo conditions on the skin, where bacteria are reported to grow as microcolonies encased in glycocalyx, the bactericidal activity of TTO against isolates grown as biofilms was also compared. Biofilms formed by MSSA and MRSA isolates were completely eradicated following exposure to 5 % TTO for 1 h. In contrast, of the biofilms formed by the nine CoNS isolates tested, only five were completely killed, although a reduction in viable count was apparent for the other four isolates. These results suggest that TTO exerts a greater bactericidal activity against biofilm-grown MRSA and MSSA isolates than against some biofilm-grown CoNS isolates.

A randomized controlled trial of tea tree oil (5%) body wash versus standard body wash to prevent colonization with methicillin-resistant Staphylococcus aureus (MRSA) in critically ill adults: research protocol

Background
Over the past ten years MRSA has become endemic in hospitals and is associated with increased healthcare costs. Critically ill patients are most at risk, in part because of the number of invasive therapies that they require in the intensive care unit (ICU). Washing with 5% tea tree oil (TTO) has been shown to be effective in removing MRSA on the skin. However, to date, no trials have evaluated the potential of TTO body wash to prevent MRSA colonization or infection. In addition, detecting MRSA by usual culture methods is slow. A faster method using a PCR assay has been developed in the laboratory, but requires evaluation in a large number of patients.

Methods/Design
This study protocol describes the design of a multicentre, phase II/III prospective open-label randomized controlled clinical trial to evaluate whether a concentration of 5% TTO is effective in preventing MRSA colonization in comparison with a standard body wash (Johnsons Baby Softwash) in the ICU. In addition we will evaluate the cost-effectiveness of TTO body wash and assess the effectiveness of the PCR assay in detecting MRSA in critically ill patients. On admission to intensive care, swabs from the nose and groin will be taken to screen for MRSA as per current practice. Patients will be randomly assigned to be washed with the standard body wash or TTO body wash. On discharge from the unit, swabs will be taken again to identify whether there is a difference in MRSA colonization between the two groups.

Discussion
If TTO body wash is found to be effective, widespread implementation of such a simple colonization prevention tool has the potential to impact on patient outcomes, healthcare resource use and patient confidence both nationally and internationally.

Trial Registration
[ISRCTN65190967]

Comparison of the cidal activity of tea tree oil and terpinen-4-ol against clinical bacterial skin isolates and human fibroblast cells

Aim: The aim of this study was to compare both the antimicrobial activity of terpinen-4-ol and tea tree oil (TTO) against clinical skin isolates of meticillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) and their toxicity against human fibroblast cells.

Changes in antibiotic susceptibility in staphylococci habituated to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia)

Aims: To investigate the effect of sub-lethal challenge with tea tree oil (TTO) on the antibiotic resistance profiles of staphylococci.

Polyopes lancifolius (Halymeniales, Rhodophyta), a new component of the Japanese marine flora introduced to Europe

Polyopes lancifolius (Harvey) S. Kawaguchi & H.W. Wang has been recorded for the first time in Europe, during the summer or 2008. A small population was discovered in the Gulf of Morbihan (northeast Atlantic, France). This is the first observation of P. lancifolius outside its native range. Vegetative and reproductive morphological features are compared with previous descriptions. rbcL sequences show no divergence from Japanese populations. Imports of Pacific oysters Crassostrea gigas (Thunberg 1793) are likely to be responsible for its accidental introduction into the Gulf of Morbihan, either directly from northwest Pacific regions or indirectly (secondary dispersal) by transfers from another European oyster farming site. The history of previous algal introductions from Japan suggests that if it becomes successfully established at Morbihan, the species is likely to spread to other European coastal areas.