SummaryEnterococcal species of bacteria are now acknowledged as leading causes of bacteraemia and other serious nosocomial infections. However, surprisingly little is known about the molecular mechanisms that promote the segregational stability of antibiotic resistance and other plasmids in these bacteria. Plasmid pRUM (24 873 bp) is a multidrug resistance plasmid identified in a clinical isolate of Enterococcus faecium . A novel proteic-based toxin-antitoxin cassette identified on pRUM was demonstrated to be a functional segregational stability module in both its native host and evolutionarily diverse bacterial species. Induced expression of the toxin protein (Txe) of this system resulted in growth inhibition in Escherichia coli . The toxic effect of Txe was alleviated by coexpression of the antitoxin protein, Axe. Homologues of the axe and txe genes are present in the genomes of a diversity of Eubacteria. These homologues ( yefMyoeB ) present in the E. coli chromosome function as a toxin-antitoxin mechanism, although the Axe and YefM antitoxin components demonstrate specificity for their cognate toxin proteins in vivo . Axe-Txe is one of the first functional proteic toxin-antitoxin systems to be accurately described for Gram-positive bacteria.

Effective assessment of laboratory practicals is a challenge for large-size classes. To reduce the administrative burden of staff members without compromising the student learning experience, we utilized dedicated computer software for short-answer question assessment for nearly 300 students and compared it with the more traditional, paper-based method of assessment of the same student cohort. Students were generally favorably disposed toward computer-assisted assessment (CAA): 75% of the students responded that for future assignments, they either had no preference for the method of assessment or would prefer CAA. Advantages were perceived to be remote access to the questions and ease of submission. The most common disadvantage cited was lack of internet access. Various advantages of CAA were mentioned by staff members: notably, the reduction in marking time and reduction of paperwork as well as the potential for the software to detect plagiarism and to administer anonymous marking. Disadvantages to CAA were the need to tailor questions to the technology, having to adapt to reading answers and marking onscreen, and the quality of feedback to students. All of the disadvantages could be overcome by training and improved versions of CAA software, currently under development. The use of CAA has proved to be a welcome addition to the tools available to staff members for the assessment of practical classes, and future improved versions of the software will increase the utility of this assessment method.

Genotypes and putative genetic relationships were characterised for epidemic methicillin-resistant Staphylococcus aureus (EMRSA) strains and isolates from England and Wales, using a high resolution DNA fingerprinting technique, fluorescent amplified-fragment length polymorphism (FAFLP). Each of the phage types of EMRSA had a distinct FAFLP profile. The technique revealed clusters of strains and isolates, and could distinguish isolates belonging to the same phage type. FAFLP provides a new approach to the epidemiological study and control of MRSA.

A representative panel of 50 European MRSA isolates was subjected to genotype analysis by¯uorescent ampli®ed-fragment length polymorphism (FAFLP) and by macrorestriction pulsed-®eld gel electrophoresis (PFGE). Each isolate had a unique pro®le with FAFLP. To model genetic relationships within the continuing MRSA epidemic, cluster analysis of FAFLP data was made, revealing nine clone complexes of MRSA. Most of these were also found by PFGE. A number of isolates had FAFLP pro®les signi®cantly different from others, and might represent emerging epidemic strains. FAFLP analysis proved particularly suitable for surveillance of the MRSA epidemic at national and international levels.

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was investigated for its ability to identify and subtype isolates of an epidemic methicillin-resistant phage type of Staphylococcus aureus, EMRSA-15. These isolates were also characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) of the coagulase gene and pulsed-field gel electrophoresis (PFGE). For FAFLP, DNA was double digested with restriction enzymes ApaI plusTaqI or EcoRI plus MseI. Site-specific adaptors were ligated to one or the other set of restriction fragments, and PCR amplification was carried out with adaptor-specific primers. Amplified fragments separated on an ABI 377 automated sequencer and analyzed with Genescan version 2.1 software generated FAFLP profiles for all the isolates. The presence or absence of fragments was scored, similarity coefficients were calculated, and UPGMA (unweighted pair group method using arithmatic averages) cluster analysis was performed. Either enzyme-primer combination readily differentiated EMRSA-15 from other methicillin-resistant S. aureus (MRSA) isolates and also revealed heterogeneity within the phage type. The discriminatory power of FAFLP was high. By combining both enzyme-primer data sets, 24 isolates were divided into 11 profiles. PCR-RFLP did not discriminate among these EMRSA-15 isolates. PFGE could discriminate well between isolates but was not as reproducible as FAFLP. All S. aureus and MRSA isolates in this study were typeable by FAFLP, which was easy to perform, robust, and reproducible, with evident potential to subtype MRSA for purposes of hospital infection control.