Recurrent microbial keratitis and endogenous site Staphylococcus aureus colonisation.

This study investigated Staphylococcus aureus carriage in patients with microbial keratitis (MK). 215 patients with MK, 60 healthy controls and 35 patients with rheumatoid arthritis (RA) were included. Corneal scrapes were collected from patients with MK. Conjunctival, nasal and throat swabs were collected from the non-MK groups on a single occasion and from the MK group at presentation and then at 6 and 12 weeks. Samples were processed using conventional diagnostic culture. 68 (31.6%) episodes of clinically suspected MK were classed as recurrent. Patients with recurrent MK had a higher isolation rate of S. aureus from their cornea than those with a single episode (p < 0.01) and a higher isolation rate of S. aureus from their conjunctiva compared to control participants, 20.6% (14/68) versus 3% (5/60) respectively (p = 0.01). Significantly more patients with recurrent MK (12/68, 17.6%) were found to have S. aureus isolated from both their conjunctiva and nose than those with a single episode of MK (7/147, 4.8% p = 0.002) and compared to patients in the control group (3/60, 5.0% p = 0.03). The results indicate that patients with recurrent MK have higher rates of carriage of S. aureus suggesting endogenous site colonisation as a possible source of recurrent infection.

Dual transcriptional-translational cascade permits cellular level tuneable expression control.

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.

lukSF-PV in Staphylococcus aureus keratitis isolates and association with clinical outcome.

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-PV+ve, mecA+ve, and lukSF-PV/mecA-ve. RESULTS: Of 95 isolates, 9 (9.5%) were lukSF-PV+ve, 9 (9.5%) mecA+ve, 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-PV+ve isolates to the antimicrobials tested, except for tigecycline (P < 0.05). The mecA+ve 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-PV+ve group (P < 0.05). The proportion of patients, however, who required surgery was significantly greater among patients with lukSF-PV+ve isolates with an odds ratio of 7.8 (95% CI 1-42, P = 0.018) for patients requiring surgery. CONCLUSIONS: lukSF-PV+ve 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.

Initial treatment of Pseudomonas aeruginosa contact lens-associated keratitis with topical chloramphenicol, and effect on outcome.

AIM: To determine whether initial empiric treatment of cases with Pseudomonas aeruginosa contact lens-associated keratitis (CLAK) with chloramphenicol had an adverse effect on outcome. METHODS: We retrospectively reviewed 139 cases of culture-proven P. aeruginosa CLAK seen between 2007 and 2009. We recorded chloramphenicol use prior to the prescription of a fluoroquinolone, the visual acuity (VA) when the fluoroquinolone was started and at final follow-up, complications and duration of follow-up. RESULTS: 46 patients (33.1%) had used chloramphenicol before they were prescribed a fluoroquinolone. When we compared this group with patients who had initial treatment with a fluoroquinolone, the ulcer size was larger when a fluoroquinolone was started (Mann-Whitney, p=0.018). Although the initial VA was also worse in the chloramphenicol group (p=0.02), and complications more frequent (p=0.016), the final VA in both groups was similar (p=0.29). The chloramphenicol group had a longer median follow-up of 37 days (IQR: 9-310 days) compared with 21 days (IQR: 6-80 days) for the non-chloramphenicol group (p=0.09). CONCLUSIONS: 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.

Genotypic analysis of UK keratitis-associated Pseudomonas aeruginosa suggests adaptation to environmental water as a key component in the development of eye infections.

To examine temporal dynamics of corneal infection (keratitis)-associated Pseudomonas aeruginosa, we compared the genetic characteristics of isolates collected during two different time periods (2003-2004 and 2009-2010) using an ArrayTube genotyping system. The distribution of keratitis-associated isolates from the two studies (n = 123) among a database of P. aeruginosa strains of non-ocular origin (n = 322) indicated that 71% of UK keratitis-associated P. aeruginosa isolates clustered together, and there was no evidence for major variations in the distribution of clone types between the two collections. Our analysis indicates the presence of a 'core keratitis cluster', associated with corneal infections, that is related to the P. aeruginosa eccB clonal complex, which is associated with adaptation to survival in environmental water. This suggests that adaptation to environmental water is a key factor in the ability of P. aeruginosa to cause eye infections.

SalB inactivation modulates culture supernatant exoproteins and affects autolysis and viability in Enterococcus faecalis OG1RF.

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.

The Enterococcus faecalis exoproteome: identification and temporal regulation by Fsr.

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.

Comparative genomics and transduction potential of Enterococcus faecalis temperate bacteriophages.

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, PhiFL1 to 3, were found to be sequence related, with PhiFL1A to C and PhiFL2A and B sharing the greatest identity (87 to 88%), while PhiFL3A and B share 37 to 41% identity with PhiFL1 and 2. PhiFL4A shares 3 to 12% identity with the phages PhiFL1 to 3. The PhiFL3A 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.