Phenotypic and Genotypic Characterization of Novel Polyvalent Bacteriophages With Potent In Vitro Activity Against an International Collection of Genetically Diverse Staphylococcus aureus.

Phage therapy recently passed a key milestone with success of the first regulated clinical trial using systemic administration. In this single-arm non-comparative safety study, phages were administered intravenously to patients with invasive Staphylococcus aureus infections with no adverse reactions reported. Here, we examined features of 78 lytic S. aureus phages, most of which were propagated using a S. carnosus host modified to be broadly susceptible to staphylococcal phage infection. Use of this host eliminates the threat of contamination with staphylococcal prophage - the main vector of S. aureus horizontal gene transfer. We determined the host range of these phages against an international collection of 185 S. aureus isolates with 56 different multilocus sequence types that included multiple representatives of all epidemic MRSA and MSSA clonal complexes. Forty of our 78 phages were able to infect > 90% of study isolates, 15 were able to infect > 95%, and two could infect all 184 clinical isolates, but not a phage-resistant mutant generated in a previous study. We selected the 10 phages with the widest host range for in vitro characterization by planktonic culture time-kill analysis against four isolates:- modified S. carnosus strain TM300H, methicillin-sensitive isolates D329 and 15981, and MRSA isolate 252. Six of these 10 phages were able to rapidly kill, reducing cell numbers of at least three isolates. The four best-performing phages, in this assay, were further shown to be highly effective in reducing 48 h biofilms on polystyrene formed by eight ST22 and eight ST36 MRSA isolates. Genomes of 22 of the widest host-range phages showed they belonged to the Twortvirinae subfamily of the order Caudovirales in three main groups corresponding to Silviavirus, and two distinct groups of Kayvirus. These genomes assembled as single-linear dsDNAs with an average length of 140 kb and a GC content of c. 30%. Phages that could infect > 96% of S. aureus isolates were found in all three groups, and these have great potential as therapeutic candidates if, in future studies, they can be formulated to maximize their efficacy and eliminate emergence of phage resistance by using appropriate combinations.

Methylcellulose Hydrogel with Melissa officinalis Essential Oil as a Potential Treatment for Oral Candidiasis.

Candida spp. are the most prevalent fungi of the human microbiota and are opportunistic pathogens that can cause oral candidiasis. Management of such infections is limited due to the low number of antifungal drugs available, their relatively high toxicity and the emergence of antifungal resistance. Therefore, much interest in the antimicrobial potential of natural compounds has recently been evident. The use of hydrogels in the delivery of biocides has been explored due to their biocompatibility, ease with drug encapsulation, and due to their potential to confer mechanical and structural properties similar to biological tissue. Methylcellulose hydrogels (10% (w/v)) with 1% (v/v) and 2% (v/v) Melissa officinalis oil were synthesised. The rheological properties and gelation time of the hydrogels were evaluated. Antimicrobial action, the antifungal potential and ability to displace Candida were determined. Rheological tests revealed that the hydrogel jellified in three minutes at 37 °C. Loaded hydrogels successfully inhibited Candida albicans growth as evident by zone of inhibition and time-kill assays. A significant reduction in retained C. albicans was demonstrated with the hydrogel at 2% Melissa officinalis concentration. This work demonstrated that an essential oil-loaded hydrogel had the potential to provide a novel antimicrobial therapy for the treatment of oral candidiasis.

Zeolite-embedded silver extends antimicrobial activity of dental acrylics.

The insertion of prosthetic devices into the oral cavity affects the oral microflora and results in accumulation of microorganisms on the prosthetic surface. Such fouling of denture surfaces can lead to a number of oral diseases and consequently to the replacement of the denture. Here, we report the post-synthesis introduction of silver in zeolite-loaded dental acrylic (DAZ) resins that does not influence the mechanical or aesthetic properties of the DA resins, and provides them with a long-term antimicrobial activity. Na-FAU zeolite (2 wt%) was incorporated into DA resin, which was conventionally processed and cut into 10 mm × 20 mm × 3 mm coupons. The Na+ in the zeolite was then exchanged with Ag+ via immersion of the DAZ coupons in 0.01 M AgNO3 solution to obtain DAZ/Ag-treated coupons used in antimicrobial tests. Antimicrobial tests showed that the DAZ/Ag-treated coupons were active against Candida albicans (a reference and a clinically relevant strain), Streptococcus mutans and Fusobacterium nucleatum. Ag leaching tests on the Ag-charged coupons at 1, 2, 3, 4, 7, 14, 30 and 45 days of incubation in distilled water at 37 °C, indicated sustained release of silver. Antimicrobial tests using a reference Candida albicans strain showed that the leached coupons retained antimicrobial activity after 45 days immersion in distilled water, but, after 60 days incubation no antimicrobial activity was observed. Cytotoxicity assay results indicated that the DAZ/Ag-treated coupons showed no additional cytotoxicity compared to neat dental acrylic coupons.

Antifungal Activity of Commercial Essential Oils and Biocides against Candida Albicans.

Management of oral candidosis, most frequently caused by Candida albicans, is limited due to the relatively low number of antifungal drugs and the emergence of antifungal tolerance. In this study, the antifungal activity of a range of commercial essential oils, two terpenes, chlorhexidine and triclosan was evaluated against C. albicans in planktonic and biofilm form. In addition, cytotoxicity of the most promising compounds was assessed using murine fibroblasts and expressed as half maximal inhibitory concentrations (IC50). Antifungal activity was determined using a broth microdilution assay. The minimum inhibitory concentration (MIC) was established against planktonic cells cultured in a range of concentrations of the test agents. The minimal biofilm eradication concentration (MBEC) was determined by measuring re-growth of cells after pre-formed biofilm was treated for 24 h with the test agents. All tested commercial essential oils demonstrated anticandidal activity (MICs from 0.06% (v/v) to 0.4% (v/v)) against planktonic cultures, with a noticeable increase in resistance exhibited by biofilms (MBECs > 1.5% (v/v)). The IC50s of the commercial essential oils were lower than the MICs, while a one hour application of chlorhexidine was not cytotoxic at concentrations lower than the MIC. In conclusion, the tested commercial essential oils exhibit potential as therapeutic agents against C. albicans, although host cell cytotoxicity is a consideration when developing these new treatments.

Novel Nystatin A1 derivatives exhibiting low host cell toxicity and antifungal activity in an in vitro model of oral candidosis.

Opportunistic oral infections caused by Candida albicans are frequent problems in immunocompromised patients. Management of such infections is limited due to the low number of antifungal drugs available, their relatively high toxicity and the emergence of antifungal resistance. Given these issues, our investigations have focused on novel derivatives of the antifungal antibiotic Nystatin A1, generated by modifications at the amino group of this molecule. The aims of this study were to evaluate the antifungal effectiveness and host cell toxicity of these new compounds using an in vitro model of oral candidosis based on a reconstituted human oral epithelium (RHOE). Initial studies employing broth microdilution, revealed that against planktonic C. albicans, Nystatin A1 had lower minimal inhibitory concentration than novel derivatives. However, Nystatin A1 was also markedly more toxic against human keratinocyte cells. Interestingly, using live/dead staining to assess C. albicans and tissue cell viability after RHOE infection, Nystatin A1 derivatives were more active against Candida with lower toxicity to epithelial cells than the parent drug. Lactate dehydrogenase activity released by the RHOE indicated a fourfold reduction in tissue damage when certain Nystatin derivatives were used compared with Nystatin A1. Furthermore, compared with Nystatin A1, colonisation of the oral epithelium by C. albicans was notably reduced by the new polyenes. In the absence of antifungal agents, confocal laser scanning microscopy showed that C. albicans extensively invaded the RHOE. However, the presence of the novel derivatives greatly reduced or totally prevented this fungal invasion.

Biocide activity against urinary catheter pathogens.

Antimicrobial effects of essential oils against bacteria associated with urinary catheter infection was assessed. Tests were performed on 14 different bacterial species cultured either planktonically or as biofilms. Biofilms were found to be up to 8-fold more tolerant of the test agents. Higher antimicrobial tolerance was also evident in tests conducted in artificial urine. Eugenol exhibited higher antimicrobial effects against both planktonic cells and biofilms than did terpinen, tea tree oil, and cineole.

Development of an "early warning" sensor for encrustation of urinary catheters following Proteus infection.

Biofilm formation in long-term urinary catheterized patients can lead to encrustation and blockage of urinary catheters with serious clinical complication. Catheter encrustation stems from infection with urease-producing bacteria, particularly Proteus mirabilis. Urease generates ammonia from urea, and the elevated pH of the urine results in crystallization of calcium and magnesium phosphates, which block the flow of urine. The aim of this research is to develop an "early warning" silicone sensor for catheter encrustation following bacterial infection of an in vitro bladder model system. The in vitro bladder model was infected with a range of urease positive and negative bacterial strains. Developed sensors enabled catheter blockage to be predicted ~17-24 h in advance of its occurrence. Signaling only occurred following infection with urease positive bacteria and only when catheter blockage followed. In summary, sensors were developed that could predict urinary catheter blockage in in vitro infection models. Translation of these sensors to a clinical environment will allow the timely and appropriate management of catheter blockage in long-term catheterized patients.

Gynecologic oncology training systems in Europe: a report from the European network of young gynaecological oncologists.

OBJECTIVE: The objectives of the study were to highlight some of the differences in training systems and opportunities for training in gynecologic oncology across Europe and to draw attention to steps that can be taken to improve training prospects and experiences of European trainees in gynecologic oncology. METHODS: The European Network of Young Gynaecological Oncologists national representatives from 34 countries were asked to review and summarize the training system in their countries of origin and fulfill a mini-questionnaire evaluating different aspects of training. We report analysis of outcomes of the mini-questionnaire and subsequent discussion at the European Network of Young Gynaecological Oncologists national representatives Asian Pacific Organization for Cancer Prevention meeting in Istanbul (April 2010). RESULTS: Training fellowships in gynecologic oncology are offered by 18 countries (53%). The median duration of training is 2.5 years (interquartile range, 2.0-3.0 years). Chemotherapy administration is part of training in 70.5% (24/34) countries. Most of the countries (26/34) do not have a dedicated national gynecologic-oncology journal. All trainees reported some or good access to training in advanced laparoscopic surgical techniques, whereas 41% indicated no access, and 59% some access to training opportunities in robotic surgery. European countries were grouped into 3 different categories on the basis of available training opportunities in gynecologic oncology: well-structured, moderately structured, and loosely structured training systems. CONCLUSIONS: There is a need for further harmonization and standardization of training programs and structures in gynecologic oncology across Europe. This is of particular relevance for loosely structured countries that lag behind the moderately structured and well-structured ones.

Antimicrobial tolerance and the significance of persister cells in recalcitrant chronic wound biofilms.

The application of antimicrobials in the management of wounds is a complex procedure requiring appropriate clinical decision making, judgment and a thorough understanding of antimicrobial therapies, together with their potential disadvantages. There is considerable direct and indirect evidence for the presence of bacterial biofilms in the chronic wound bed, and it has been demonstrated that bacteria within these biofilms may exhibit both specific and nonspecific antimicrobial tolerance. The antimicrobial tolerance of biofilms is a major concern in the treatment of both infected and nonhealing chronic wounds and an understanding of the mechanisms involved is of fundamental importance in managing wound infections and developing future wound management strategies. The aim of this review is therefore to provide an overview of our current understanding of the mechanisms by which bacteria in wound biofilms can resist conventional antibiotic and antibacterial therapies which is very important to wound healing.

An in vitro model of chronic wound biofilms to test wound dressings and assess antimicrobial susceptibilities.

OBJECTIVES: The targeted disruption of biofilms in chronic wounds is an important treatment strategy and the subject of intense research. In the present study, an in vitro model of chronic wound biofilms was developed to assess the efficacy of antimicrobial treatments for use in the wound environment. METHODS: Using chronic wound isolates, assays of bacterial coaggregation established that aerobic and anaerobic wound bacteria were able to coaggregate and form biofilms. A constant depth film fermenter (CDFF) was used to develop wound biofilms in vitro, which were analysed using light microscopy and scanning electron microscopy. The susceptibility of bacteria within these biofilms was examined in response to the most frequently prescribed 'chronic wound' antibiotics and a series of iodine- and silver-containing commercial antimicrobial products and lactoferrin. RESULTS: Defined biofilms were rapidly established within 1-2 days. Antibiotic treatment demonstrated that mixed Pseudomonas and Staphylococcus biofilms were not affected by ciprofloxacin (5 mg/L) or flucloxacillin (15 mg/L), even at concentrations equivalent to twice the observed peak serum levels. The results contrasted with the ability of povidone-iodine (1%) to disrupt the wound biofilm; an effect that was particularly pronounced in the dressing testing where iodine-based dressings completely disrupted established 7 day biofilms. In contrast, only two of six silver-containing dressings exhibited any effect on 3 day biofilms, with no effect on 7 day biofilms. CONCLUSIONS: This wound model emphasizes the potential role of the biofilm phenotype in the observed resistance to antibiotic therapies that may occur in chronic wounds in vivo.

Detection and identification of specific bacteria in wound biofilms using peptide nucleic acid fluorescent in situ hybridization (PNA FISH).

Biofilms provide a reservoir of potentially infectious micro-organisms that are resistant to antimicrobial agents, and their importance in the failure of medical devices and chronic inflammatory conditions is increasingly being recognized. Particular research interest exists in the association of biofilms with wound infection and non-healing, i.e. chronic wounds. In this study, fluorescent in situ hybridization (FISH) was used in combination with confocal laser scanning microscopy (CLSM) to detect and characterize the spatial distribution of biofilm-forming bacteria which predominate within human chronic skin wounds (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sp. and Micrococcus sp.). In vitro biofilms were prepared using a constant-depth film fermenter and a reconstituted human epidermis model. In vivo biofilms were also studied using biopsy samples from non-infected chronic venous leg ulcers. The specificity of peptide nucleic acid (PNA) probes for the target organisms was confirmed using mixed preparations of planktonic bacteria and multiplex PNA probing. Identification and location of individual bacterial species within multi-species biofilms demonstrated that P. aeruginosa was predominant. CLSM revealed clustering of individual species within mixed-species biofilms. FISH analysis of archive chronic wound biopsy sections showed bacterial presence and allowed bacterial load to be determined. The application of this standardized procedure makes available an assay for identification of single- or multi-species bacterial populations in tissue biopsies. The technique provides a reliable tool to study bacterial biofilm formation and offers an approach to assess targeted biofilm disruption strategies in vivo.

Characterization of Candida parapsilosis infection of an in vitro reconstituted human oral epithelium.

Oral candidosis is a common problem in immunocompromised patients, and whilst Candida albicans is regarded as the principal cause of infection, other non-Candida albicans Candida (NCAC) species are increasingly being recognized as human pathogens. Relatively little is known about the virulence factors associated with NCAC species, and the aim of this study was to use a reconstituted human oral epithelium (RHOE) to examine epithelial infection withCandida parapsilosis. Strains originating from the oral and vaginal mucosa and from the urinary tract were all shown to colonize RHOE in a strain-dependent manner. Strain differences were found in the colonizing morphology and in the extent of invasion of the RHOE. Low invasion of RHOE was detected for strains after 12 h, whereas extensive tissue damage was evident after 24 h when assessed using histological examination and lactate dehydrogenase activity determination. Tissue damage was reduced in the presence of pepstatin A, although C. parapsilosis invasion of the tissue was not inhibited. Real-time polymerase chain reaction of secreted aspartyl proteinase (SAP) genes (SAPP1-3) showed that expression was strain dependent, with an increased expression generally occurring for Candida infecting RHOE compared with planktonic equivalents. In summary, C. parapsilosis was not highly invasive of RHOE but did induce significant tissue damage, which could relate to specific SAPgene expression.