Karen Smith

Comparison of biofilm-associated cell survival following in vitro exposure of meticillin-resistant Staphylococcus aureus biofilms to the antibiotics clindamycin, daptomycin, linezolid, tigecycline and vancomycin

The efficacy of commonly used antistaphylococcal antimicrobials (clindamycin, linezolid and vancomycin) and recently developed antibiotics (daptomycin and tigecycline) was compared against clinical isolates of meticillin-resistant Staphylococcus aureus (MRSA). Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations, time-kill kinetics and biofilm-associated cell survival were examined for 12 clinical isolates of MRSA treated with each antibiotic. The MIC ranges for daptomycin, linezolid, tigecycline, clindamycin and vancomycin were 0.06-0.25, 1-2, 0.06, 0.125-1024 and 0.5-1 microg/mL, respectively. Daptomycin and vancomycin were bactericidal following 6h of incubation with planktonic cells, whilst clindamycin, linezolid and tigecycline were bacteriostatic. None of the antibiotics killed 100% of biofilm-associated cells. Mean cell survival in biofilms treated with clindamycin, daptomycin, linezolid, tigecycline and vancomycin was 62%, 4%, 45%, 43% and 19%, respectively. Although all antibiotics were effective against planktonic staphylococcal populations, vancomycin and daptomycin possessed superior activity against biofilm-associated cells.

Biofilm formation by Scottish clinical isolates of Staphylococcus aureus

The biofilm-forming capacity of 972 clinical isolates of Staphylococcus aureus was tested using a high-throughput polystyrene 96-peg plate format. Isolates of S. aureus were collected from patients in hospitals throughout Scotland from 2004 to 2006; 763 of these were meticillin-resistant S. aureus (MRSA) and 209 were meticillin-sensitive S. aureus (MSSA). The biomass of each biofilm was quantified using a crystal violet staining technique. Isolates were divided into those that formed fully established biofilms, moderately attached biofilms and weakly adherent biofilms by comparison with a known biofilm-forming strain. The majority of MRSA (53.8 %) and MSSA (43.5 %) isolates formed moderately attached biofilms. Fully established biofilms were formed by 20.5 % of MRSA isolates and 28.0 % of MSSA isolates, whilst 25.7 % of MRSA isolates and 28.5 % of MSSA isolates formed negligible biofilms. There was no significant correlation between susceptibility to meticillin and biofilm formation (P=0.77). MRSA isolates were divided into clonal types (EMRSA-15, EMRSA-16 and sporadic isolates) based on PFGE genotyping results. EMRSA-15 isolates formed significantly more moderately and fully established biofilms than EMRSA-16 isolates (P<0.001). S. aureus strains isolated from the skin of patients had a significantly greater capacity to form biofilms than isolates from other body sites, including the blood. Microscopic examination of biofilms by scanning electron microscopy (SEM) revealed that poorly adherent biofilm formers failed to colonize the entire surface of the peg, whilst moderately adherent biofilm formers grew in uniform monolayers but failed to develop a mature three-dimensional structure. SEM analysis of an isolate representative of the group that formed fully established biofilms confirmed that this isolate developed a dense biofilm with a textured, multi-layered, three-dimensional structure.

Transnational teaching experiences : an under‐explored territory for transformative professional development

In an increasingly global higher education environment, many universities are taking their educational services overseas. One model of overseas provision is through transnational (offshore) education. Transnational teaching challenges the prevailing understanding of an academic role at every level. Transnational teachers are expected to work in environments, climates and classrooms which are culturally very different to their own. Assumptions about university education are shaken and teachers find themselves having to return to and question the fundamentals of their teaching, learning and assessment practices. This paper argues that the experience of being a transnational teacher and working in a culture very different to one’s own forces reflection which can lead to ‘perspective transformation’; as such it could be a powerful professional development opportunity which should be nurtured and supported. This paper draws on the growing body of published literature on staff experiences of transnational teaching and my own experiences as a transnational teacher to support this argument. It shows that the ‘novel experiences’ of transnational teaching encourage content, process and premise reflection that can, with appropriate support, ultimately improve teaching practice not just in the transnational context, but also back home. Dans un environnement de l’enseignement supérieur de plus en plus globalisé, nombre d’universités étendent leur prestation de services éducatifs outremer. L’éducation transnationale délocalisée (offshore education) est un de ces modèles de prestation de services outremer. L’enseignement transnational pose des défis à la compréhension dominante du rôle académique à tous les niveaux. Les présupposés au sujet de l’éducation universitaire sont remis en question et les enseignants doivent dès lors revoir et remettre en question les fondements de leurs pratiques liées à l’enseignement, à l’apprentissage et à l’évaluation. Cet article défend l’idée que l’expérience de devenir un enseignant transnational et d’œuvrer dans une culture qui est très différente de la sienne force une réflexion qui peut conduire à une « transformation de perspective ». En ce sens, il pourrait s’agir d’une occasion de développement professionnel puissante, laquelle devrait être soutenue et encouragée. L’article repose sur un corpus de documentation en pleine croissance portant sur l’enseignement transnational ainsi que sur mes propres expériences à titre d’enseignement transnational. Il fait la démonstration que des « expériences nouvelles » de l’enseignement transnational encouragent une réflexion au sujet du contenu, du processus et des présupposés, laquelle, en fonction d’un soutien adapté, peut en fin de compte améliorer la pratique enseignante non seulement dans le contexte transnational mais aussi à domicile.

Biofilms formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity

BackgroundCandida albicans infections have become increasingly recognised as being biofilm related. Recent studies have shown that there is a relationship between biofilm formation and poor clinical outcomes in patients infected with biofilm proficient strains. Here we have investigated a panel of clinical isolates in an attempt to evaluate their phenotypic and transcriptional properties in an attempt to differentiate and define levels of biofilm formation.ResultsBiofilm formation was shown to be heterogeneous; with isolates being defined as either high or low biofilm formers (LBF and HBF) based on different biomass quantification. These categories could also be differentiated using a cell surface hydrophobicity assay with 24xa0h biofilms. HBF isolates were more resistance to amphotericin B (AMB) treatment than LBF, but not voriconazole (VRZ). In a Galleria mellonella model of infection HBF mortality was significantly increased in comparison to LBF. Histological analysis of the HBF showed hyphal elements intertwined indicative of the biofilm phenotype. Transcriptional analysis of 23 genes implicated in biofilm formation showed no significant differential expression profiles between LBF and HBF, except for Cdr1 at 4 and 24xa0h. Cluster analysis showed similar patterns of expression for different functional classes of genes, though correlation analysis of the 4xa0h biofilms with overall biomass at 24xa0h showed that 7 genes were correlated with high levels of biofilm, including Als3, Eap1, Cph1, Sap5, Plb1, Cdr1 and Zap1.ConclusionsOur findings show that biofilm formation is variable amongst C. albicans isolates, and categorising isolates depending on this can be used to predict how pathogenic the isolate will behave clinically. We have shown that looking at individual genes in less informative than looking at multiple genes when trying to categorise isolates at LBF or HBF. These findings are important when developing biofilm-specific diagnostics as these could be used to predict how best to treat patients infected with C. albicans. Further studies are required to evaluate this clinically.

Assuring quality in transnational higher education: a matter of collaboration or control?

As transnational education collaborations increase so too do concerns about the quality of provision. To address these concerns, national codes of good practice have been written to guide exporting institutions on how to set up and manage international collaborations, so that academic standards and student experiences are not compromised. This article attempts to show how linguistic analysis can be used to decode underlying messages in the way we describe quality assurance processes. It looks at three codes of practice from major exporters of higher education: USA, UK and Australia. The article focuses specifically on the roles and responsibilities of the awarding higher education institution and their partner, issues of equivalence and opportunities for adaptation of curricula to meet global and local requirements. The analysis raises questions about the portrayal of transnational higher education within the documents.

Dentures are a Reservoir for Respiratory Pathogens

PURPOSEnRecent studies have established a relationship between dental plaque and pulmonary infection, particularly in elderly individuals. Given that approximately one in five adults in the UK currently wears a denture, there remains a gap in our understanding of the direct implications of denture plaque on systemic health. The aim of this study was to undertake a comprehensive evaluation of putative respiratory pathogens residing upon dentures using a targeted quantitative molecular approach.nnnMATERIALS AND METHODSnOne hundred and thirty patients dentures were sonicated to remove denture plaque biofilm from the surface. DNA was extracted from the samples and was assessed for the presence of respiratory pathogens by quantitative polymerase chain reaction (qPCR). Ct values were then used to approximate the number of corresponding colony forming equivalents (CFEs) based on standard curves.nnnRESULTSnOf the dentures, 64.6% were colonized by known respiratory pathogens. Six species were identified: Streptococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae B, Streptococcus pyogenes, and Moraxella catarrhalis. P. aeruginosa was the most abundant species followed by S. pneumoniae and S. aureus in terms of average CFE and overall proportion of denture plaque. Of the participants, 37% suffered from denture stomatitis; however, there were no significant differences in the prevalence of respiratory pathogens on dentures between healthy and inflamed mouths.nnnCONCLUSIONSnOur findings indicate that dentures can act as a reservoir for potential respiratory pathogens in the oral cavity, thus increasing the theoretical risk of developing aspiration pneumonia. Implementation of routine denture hygiene practices could help to reduce the risk of respiratory infection among the elderly population.

One step closer to understanding the role of bacteria in diabetic foot ulcers: characterising the microbiome of ulcers

BackgroundThe aim of this study was to characterise the microbiome of new and recurrent diabetic foot ulcers using 16S amplicon sequencing (16S AS), allowing the identification of a wider range of bacterial species that may be important in the development of chronicity in these debilitating wounds. Twenty patients not receiving antibiotics for the past three months were selected, with swabs taken from each individual for culture and 16S AS. DNA was isolated using a combination of bead beating and kit extraction. Samples were sequenced on the Illumina Hiseq 2500 platform.ResultsConventional laboratory culture showed positive growth from only 55xa0% of the patients, whereas 16S AS was positive for 75xa0% of the patients (41 unique genera, representing 82 different operational taxonomic units (OTU’s). S. aureus was isolated in 72xa0% of culture-positive samples, whereas the most commonly detected bacteria in all ulcers were Peptoniphilus spp., Anaerococcus spp. and Corynebacterium spp., with the addition of Staphylococcus spp. in new ulcers. The majority of OTU’s residing in both new and recurrent ulcers (over 67xa0%) were identified as facultative or strict anaerobic Gram-positive organisms. Principal component analysis (PCA) showed no difference in clustering between the two groups (new and recurrent ulcers).ConclusionsThe abundance of anaerobic bacteria has important implications for treatment as it suggests that the microbiome of each ulcer “starts afresh” and that, although diverse, are not distinctly different from one another with respect to new or recurrent ulcers. Therefore, when considering antibiotic therapy the duration of current ulceration may be a more important consideration than a history of healed ulcer.

Extracellular DNA release confers heterogeneity in Candida albicans biofilm formation.

BackgroundBiofilm formation by Candida albicans has shown to be highly variable and is directly associated with pathogenicity and poor clinical outcomes in patients at risk. The aim of this study was to test the hypotheses that the extracellular DNA release by C. albicans is strain dependent and is associated with biofilm heterogeneity.ResultsInitially, biofilm formed by C. albicans high biofilm formers (HBF) or low biofilm formers (LBF) were treated with DNase to find whether eDNA play a role in their biofilm formation. Digestion of biofilm eDNA significantly reduced the HBF biofilm biomass by five fold compared to untreated controls. In addition, quantification of eDNA over the period of biofilm formation by SYBR green assay demonstrate a significantly higher level of 2 to 6 fold in HBF compared to LBF. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase genes, a marker of autolysis, were upregulated in 24xa0h biofilm formation by HBF compared to LBF, indicating autolysis pathway possibly involved in causing variation. The biofilm biomass and eDNA release by single (∆cht2, ∆cht3) and double knockout (∆cht2/∆cht3) chitinase mutants were significantly less compared to their parental strain CA14, confirming the role of chitinases in eDNA release and biofilm formation. Correlation analysis found a positive correlation between chitinases and HWP 1, suggesting eDNA may release during the hyphal growth. Finally, we showed a combinational treatment of biofilms with DNase or chitinase inhibitor (acetazolamide) plus amphotericin B significantly improved antifungal susceptibility by 2 to 8 fold.ConclusionsCollectively, these data show that eDNA release by C. albicans clinical isolates is variable and is associated with differential biofilm formation. Digestion of biofilm eDNA by DNase may provide a novel therapeutic strategies to destabilise biofilm growth and improves antifungal sensitivity.

Telavancin shows superior activity to vancomycin with multidrug-resistant Staphylococcus aureus in a range of in vitro biofilm models

The activity of telavancin was compared with vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) in planktonic culture and biofilms grown using a range of in vitro models. Antibiotic efficacy was determined using 24 clinical isolates, including healthcare-associated (HA)-MRSA, community-associated (CA)-MRSA and isolates with reduced (intermediate) susceptibility to vancomycin (VISA). Activity against biofilms was compared using three models: 96-peg plates, 96-well flat-bottom plates and a flow-cell system. Cell death was evaluated using a metabolic dye and Live/Dead staining. The planktonic minimum inhibitory concentration (MIC) range for telavancin was lower than that for vancomycin (0.06–0.25xa0mg/l and 0.5–8xa0mg/l, respectively). Vancomycin (100u2009×u2009MIC) killed, on average, 59xa0% of cells in HA-MRSA biofilms grown on 96-peg plates, 44xa0% of cells in CA-MRSA biofilms and 26xa0% of cells in VISA biofilms. Telavancin (100u2009×u2009MIC) killed, on average, 63xa0%, 49xa0% and 41xa0% of cells, respectively. The antibiotics showed similar efficacy against MRSA biofilms but telavancin was more effective against those formed by VISA isolates. In the flow-cell system, antibiotic cell killing was enhanced with both antibiotics, killing up to 80xa0% of biofilm-associated cells. The variance in cell killing displayed when biofilms were grown using different systems highlights the importance of selecting an appropriate model for antimicrobial efficacy tests. The flow-cell system more closely reflects conditions encountered during infection and is possibly more clinically relevant than a 96-well plate system. Despite differences between the models evaluated, telavancin typically demonstrated improved efficacy over vancomycin, indicating the potential value of the agent in the treatment of biofilm-mediated infections caused by S. aureus, especially multidrug-resistant isolates.

Aspergillus fumigatus enhances elastase production in Pseudomonas aeruginosa co-cultures

In the cystic fibrosis (CF) lung the presence of bacteria and fungi in the airways promotes an inflammatory response causing progressive lung damage, ultimately leading to high rates of morbidity and mortality. We hypothesized that polymicrobial interactions play an important role in promoting airway pathogenesis. We therefore examined the interplay between the most commonly isolated bacterial CF pathogen, Pseudomonas aeruginosa, and the most prevalent filamentous fungi, Aspergillus fumigatus, to test this. Co-culture experiments showed that in the presence of A. fumigatus the production of P. aeruginosa elastase was enhanced. This was confirmed by the presence of zones of clearance on Elastin-Congo Red (ECR) agar, which was identified as elastase by mass spectrometry. When P. aeruginosa were grown in a co-culture model with mature A. fumigatus biofilms, 60% of isolates produced significantly more elastase in the presence of the filamentous fungi than in its absence (P < .05). The expression of lasB also increased when P. aeruginosa isolates PA01 and PA14 were grown in co-culture with A. fumigatus. Supernatants from co-culture experiments were also significantly toxic to a human lung epithelial cell line (19-38% cell cytotoxicity) in comparison to supernatants from P. aeruginosa only cultures (P < .0001). Here we report that P. aeruginosa cytotoxic elastase is enhanced in the presence of the filamentous fungi A. fumigatus, suggesting that this may have a role to play in the damaging pathology associated with the lung tissue in this disease. This indicates that patients who have a co-colonisation with these two organisms may have a poorer prognosis.