To determine the relative importance of temperate bacteriophage in the horizontal gene transfer of fitness and virulence determinants of Enterococcus faecalis, a panel of 47 bacteremia isolates were treated with the inducing agents mitomycin C, norfloxacin, and UV radiation. Thirty-four phages were purified from culture supernatants and discriminated using pulsed-field gel electrophoresis (PFGE) and restriction mapping. From these analyses the genomes of eight representative phages were pyrosequenced, revealing four distinct groups of phages. Three groups of phages, ⌽FL1 to 3, were found to be sequence related, with ⌽FL1A to C and ⌽FL2A and B sharing the greatest identity (87 to 88%), while ⌽FL3A and B share 37 to 41% identity with ⌽FL1 and 2. ⌽FL4A shares 3 to 12% identity with the phages ⌽FL1 to 3. The ⌽FL3A and B phages possess a high DNA sequence identity with the morphogenesis and lysis modules of Lactococcus lactis subsp. cremoris prophages. Homologs of the Streptococcus mitis platelet binding phage tail proteins, PblA and PblB, are encoded on each sequenced E. faecalis phage. Few other phage genes encoding potential virulence functions were identified, and there was little evidence of carriage of lysogenic conversion genes distal to endolysin, as has been observed with genomes of many temperate phages from the opportunist pathogens Staphylococcus aureus and Streptococcus pyogenes. E. faecalis JH2-2 lysogens were generated using the eight phages, and these were examined for their relative fitness in Galleria mellonella. Several lysogens exhibited different effects upon survival of G. mellonella compared to their isogenic parent. The eight phages were tested for their ability to package host DNA, and three were shown to be very effective for generalized transduction of naive host cells of the laboratory strains OG1RF and JH2-2.Enterococcus faecalis is a member of the natural flora of humans and colonizes the gastrointestinal and vaginal tracts and the oral cavity. In recent years it has emerged as an important opportunistic nosocomial pathogen and is a causative agent of bacteremia, infective endocarditis, and surgical wound and urinary tract infections. The accumulation of acquired antibiotic resistance determinants, in addition to its intrinsic resistance and tenacity, has given rise to the evolution of clinical isolates of E. faecalis that are therapeutically problematic (19). Greater notoriety was afforded to this species following the observed transfer of the conjugative transposon Tn1546 to Staphylococcus aureus, imparting vancomycin resistance (11). Subsequent analysis has revealed that multiple independent E. faecalis-dependent vanA transfers had occurred in the United States prior to 2007 (50). This places enterococci in an important and dynamic position within the health care system, warranting their increased study.The specific determinants that are proposed to contribute to the virulence of E. faecalis are not universally present, and expression of the cognate genes is variable (21, 37). For exa...

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems.

Analysis of the culture supernatant exoproteins produced by two PFGE clusters of high-level gentamicin and ciprofloxacin-resistant clinical isolates of Enterococcus faecalis from the UK and Ireland revealed two distinct protein profiles. This grouping distinguished OG1RF and GelE metalloprotease-expressing isolates from JH2-2 and other GelE-negative isolates. The integrity of the fsrABDC operon was found to determine the exoproteome composition, since an fsrB mutant of strain OG1RF appeared very similar to that of strain JH2-2, and complementation of the latter with the fsrABDC operon produced an OG1RF-like exoproteome. The proteins present in the supernatant fraction of OG1RF were separated using 2D gels and identified by mass spectrometry and comprised many mass and pI variants of the GelE and SprE proteases. In addition cell wall synthesis and cell division proteins were identified. An OG1RF fsrB mutant had a distinct exoprotein fraction with an absence of the Fsr-regulated proteases and was characterised by general stress and glycolytic proteins. The exoproteome of the OG1RF fsrB mutant resembles that of a divIVA mutant of E. faecalis , suggestive of a stress phenotype.

Chloramphenicol 0.5% eye drops are available in the UK without prescription. Chloramphenicol had been used in one-third of cases of P. aeruginosa CLAK prior to the use of a broad-spectrum antimicrobial, which was associated with more complications and a longer interval to resolution, but with no adverse effect on final VA.

The culture supernatant fraction of an Enterococcus faecalis gelE mutant of strain OG1RF contained elevated levels of the secreted antigen SalB. Using differential fluorescence gel electrophoresis (DIGE) the salB mutant was shown to possess a unique complement of exoproteins. Differentially abundant exoproteins were identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Stress-related proteins including DnaK, Dps family protein, SOD, and NADH peroxidase were present in greater quantity in the OG1RF salB mutant culture supernatant. Moreover, several proteins involved in cell wall synthesis and cell division, including D-Ala-D-Lac ligase and EzrA, were present in reduced quantity in OG1RF salB relative to the parent strain. The salB mutant displayed reduced viability and anomalous cell division, and these phenotypes were exacerbated in a gelE salB double mutant. An epistatic relationship between gelE and salB was not identified with respect to increased autolysis and cell morphological changes observed in the salB mutant. SalB was purified as a six-histidine-tagged protein to investigate peptidoglycan hydrolytic activity; however, activity was not evident. High-pressure liquid chromatography (HPLC) analysis of reduced muropeptides from peptidoglycan digested with mutanolysin revealed that the salB mutant and OG1RF were indistinguishable.

PURPOSE.To determine the prevalence, genetic diversity, and clinical relevance of the lukSF-PV gene, encoding the bacterial toxin Panton-Valentine leukocidin, in Staphylococcus aureus isolates from cases of bacterial keratitis in the United Kingdom.METHODS. Multiplex PCRs investigating carriage of lukSF-PV and mecA were performed on S. aureus isolates from patients. The lukSF-PV operon was sequenced to investigate its diversity, and multilocus sequence typing to test for a clonal relationship between lukSF-PV isolates. Antimicrobial minimum inhibitory concentrations (MICs) and clinical outcome data were compared for isolates characterized as lukSF-PVþve, mecAþve, and lukSF-PV/mecA-ve. RESULTS.Of 95 isolates, 9 (9.5%) were lukSF-PVþve, 9 (9.5%) mecAþve, and 1 was positive for both. Five single nucleotide polymorphisms were found in lukSF-PV genes of seven strains. There was no significant difference between the MICs of lukSF-PV/mecA-ve and lukSF-PVþve isolates to the antimicrobials tested, except for tigecycline (P < 0.05). The mecAþve isolates had significantly higher mean MICs to meropenem and fluoroquinolones (P < 0.05). There were nonsignificant trends for healing and treatment times, ulcer and scar size, and overall clinical score to be greater in the lukSF-PVþve group (P < 0.05). The proportion of patients, however, who required surgery was significantly greater among patients with lukSF-PVþve isolates with an odds ratio of 7.8 (95% CI 1-42, P ¼ 0.018) for patients requiring surgery.CONCLUSIONS. lukSF-PVþve isolates were associated with a trend to worse clinical outcome and more surgical interventions, with an effect unrelated to MICs. This suggests that lukSF-PV may be an important virulence factor in S. aureus-associated keratitis.Keywords: keratitis, Panton-Valentine leukocidin, Staphylococcus aureus keratitis T he pathogenicity of Staphylococcus aureus is mediated by a multitude of secreted virulence factors, such as bacterial surface adhesins, immune evasion proteins, and toxins. PantonValentine leukocidin (PVL) is a pore-forming toxin produced by S. aureus.1 It is a bicomponent toxin that consists of the polypeptides LukS-PV and LukF-PV. 2 The cognate LukS-PV and LukF-PV bind to neutrophils, monocytes, and macrophages, but not to lymphocytes.3 Following the binding of monomers of LukS-PV and LukF-PV, further monomers attach to the cell wall forming a heptameric structure that forms a pore in the host cell surface. 2,4 This pore formation results in leukocyte cell death and the release of inflammatory cytokines. 3 The involvement, however, of PVL in the virulence of S. aureus is equivocal and its link with clinical outcome remains uncertain. In vivo studies have produced conflicting data. Murine models of S. aureus infection have shown that the absence of PVL results in an increase in virulence, 5-8 whereas studies in rabbits indicate that the presence of PVL increases the virulence of S. aureus.9-11 These discrepancies could, however, be attributed to differences in the immunology of the models. 11 Mo...

Intraocular metastasis from cutaneous melanoma J Shankar et al 660Eye related to the passage of the metallic fragment as it passed into the eye. A number of possible mechanisms for this can be postulated. Firstly the low pressure zone immediately following a high velocity projectile would draw air in its wake down an induced pressure gradient. This could have resulted in air being drawn into the eye, although the absence of air at the entry site or related to the path between this and the presumed site of impact is against this. The second possibility is that the bubbles represent the result of cavitation induced by the passage of the solid fragment through the semi-liquid vitreous medium. 4 A low pressure region in the wake of a fast moving object can result in dissolved gases coming out of solution. Again though, it is odd that they are solely related to the final resting site of the fragment. Thirdly, a thermal or chemical reaction between the fragment and the vitreous gel could result in the liberation of free gas, and this cannot be excluded. It therefore remains uncertain as to the exact mechanism that induced this interesting phenomenon. The authors would be pleased to hear of any further cases where similar findings were observed. Neither external beam radiotherapy nor chemotherapy was successful in achieving tumour control, with these eyes ultimately being lost as a result of neovascular glaucoma. Non-ocular systemic metastatic disease was usually present at the time of ocular diagnosis. The patients tended to develop cerebral metastases and had a mean survival of 5 months from the time of development of ocular metastases. 5,7,8 The present case shows how vitrectomy may have a therapeutic role by providing functional vision and preventing neovascular glaucoma from developing. References Case reportAn 87-year-old Caucasian lady was referred with a history of floaters and progressive decrease in vision in the left eye over a 4-week period. She had previously been operated in both eyes for cataract and had good postoperative vision. She had undergone excision of a cutaneous melanoma from the right cheek 15 months earlier. This was followed by radical neck dissection and radiotherapy. Six months before her ophthalmic presentation, several metastatic lesions were excised from the back of the neck, left arm and anterior abdominal wall.On examination, the visual acuity was 6/6 in the right eye and hand movements in the affected left eye. Anterior segment examination showed bilateral, quiet pseudophakia. Numerous pigmented clumps were seen on the anterior hyaloid face. The vitreous cavity showed multiple brown cannon-ball opacities. No retinal details were discernible. B scan ultrasonography showed several echogenic points in the vitreous cavity with a flat retina. No choroidal thickening was seen.The patient underwent a diagnostic and therapeutic pars plana vitrectomy under local anaesthesia. The vitreous cavity showed a dense collection of brown cannon-ball opacities. No choroidal or retinal masses

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