Antibiotic loaded ß-tricalcium phosphate/calcium sulfate for antimicrobial potency, prevention and killing efficacy of Pseudomonas aeruginosa and Staphylococcus aureus biofilms.

This study investigated the efficacy of a biphasic synthetic ß-tricalcium phosphate/calcium sulfate (ß-TCP/CS) bone graft substitute for compatibility with vancomycin (V) in combination with tobramycin (T) or gentamicin (G) evidenced by the duration of potency and the prevention and killing efficacies of P. aeruginosa (PAO1) and S. aureus (SAP231) biofilms in in vitro assays. Antibiotic loaded ß-TCP/CS beads were compared with antibiotic loaded beads formed from a well characterized synthetic calcium sulfate (CS) bone void filler. ß-TCP/CS antibiotic loaded showed antimicrobial potency against PAO1 in a repeated Kirby-Bauer like zone of inhibition assay for 6 days compared to 8 days for CS. However, both bead types showed potency against SAP231 for 40 days. Both formulations loaded with V + T completely prevented biofilm formation (CFU below detection limits) for the 3 days of the experiment with daily fresh inoculum challenges (P < 0.001). In addition, both antibiotic loaded materials and antibiotic combinations significantly reduced the bioburden of pre-grown biofilms by between 3 and 5 logs (P < 0.001) with V + G performing slightly better against PAO1 than V + T. Our data, combined with previous data on osteogenesis suggest that antibiotic loaded ß-TCP/CS may have potential to stimulate osteogenesis through acting as a scaffold as well as simultaneously protecting against biofilm infection. Future in vivo experiments and clinical investigations are warranted to more comprehensively evaluate the use of ß-TCP/CS in the management of orthopaedic infections.

Prevention and Killing Efficacy of Carbapenem Resistant Enterobacteriaceae (CRE) and Vancomycin Resistant Enterococci (VRE) Biofilms by Antibiotic-Loaded Calcium Sulfate Beads.

Carbapenem-resistant Enterobacteriaceae (CRE) and vancomycin-resistant Enterococci (VRE) have emerged as multidrug-resistant (MDR) pathogens associated with periprosthetic joint infections (PJI). In this study, we evaluated the efficacy of antibiotic-loaded calcium sulfate beads (ALCSB) in inhibiting bacterial growth, encouraging biofilm formation and killing preformed biofilms of CRE and VRE. Three strains of Klebsiella pneumoniae (KP) and a strain of Enterococcus faecalis (EF) were used. ALCSB of 4.8-mm diameter were loaded with vancomycin (V) and gentamicin (G), V and rifampicin (R), V and tobramycin (T) or R and meropenem (M), and placed onto tryptic soy agar (TSA), spread with one of the test strains and incubated for 24 h at 37 °C. Beads were transferred daily onto fresh TSA spread plates and the zone of inhibition (ZOI) was recorded until no inhibition was observed. ALCSB containing R + M or R + V produced the most extensive ZOI up to 5 weeks. Biofilm prevention efficacy was investigated by challenging ALCSB daily with 5 × 105 CFU/mL bacterial cells and analyzing for biofilm formation at challenges 1, 2 and 3. In the biofilm killing experiments, ALCSB were added to pre-grown 3-day biofilms of KP and EF strains, which were then analyzed at days 1 and 3 post-exposure. The CFU counts and confocal images of the attached cells showed that ALCSB treatment reduced colonization and biofilm formation significantly (5-7 logs) with combinations of R + M or R + V, compared to unloaded beads. This study provides evidence that the local release of antibiotics from ALCSB may be useful in treating the biofilms of multidrug-resistant strains of CRE and VRE.

Biofilms and delayed healing - an in vitro evaluation of silver- and iodine-containing dressings and their effect on bacterial and human cells.

This study investigated whether there are differences in the ability of wound dressings to modulate certain factors known to affect wound healing. A selection of antimicrobial dressings (AQUACEL® Ag Extra™ , AQUACEL® Ag+ Extra™ , IODOFLEX™ , ACTICOAT™ 7 and PROMOGRAN PRISMA™ matrix) were tested for their effect on both bacterial bioburden and human dermal fibroblasts. Some dressings underwent further evaluation for activity against Pseudomonas aeruginosa biofilms using a colony-drip flow reactor model. The ability of in vitro biofilms to produce proteases, and the effect of PROMOGRAN PRISMA matrix on such proteases, was also investigated. All antimicrobial dressings tested reduced vegetative bacterial load; however, only PROMOGRAN PRISMA matrix was able to significantly reduce biofilm populations (P = 0·01). Additionally, PROMOGRAN PRISMA matrix was the only dressing that did not inhibit dermal fibroblast growth. All other dressings were detrimental to cell viability. In vitro biofilms of Pseudomonas aeruginosa were demonstrated as being capable of releasing bacterial proteases into their surroundings, and incubation with PROMOGRAN PRISMA matrix led to a 77% reduction in activity of such proteases (P = 0·002). The unique ability of PROMOGRAN PRISMA matrix to reduce in vitro vegetative bacteria, biofilm bacteria and bacterial proteases while still allowing dermal fibroblast proliferation may help rebalance the wound environment and reduce the occurrence of infection.

Mitotic control of human papillomavirus genome-containing cells is regulated by the function of the PDZ-binding motif of the E6 oncoprotein.

The function of a conserved PDS95/DLG1/ZO1 (PDZ) binding motif (E6 PBM) at the C-termini of E6 oncoproteins of high-risk human papillomavirus (HPV) types contributes to the development of HPV-associated malignancies. Here, using a primary human keratinocyte-based model of the high-risk HPV18 life cycle, we identify a novel link between the E6 PBM and mitotic stability. In cultures containing a mutant genome in which the E6 PBM was deleted there was an increase in the frequency of abnormal mitoses, including multinucleation, compared to cells harboring the wild type HPV18 genome. The loss of the E6 PBM was associated with a significant increase in the frequency of mitotic spindle defects associated with anaphase and telophase. Furthermore, cells carrying this mutant genome had increased chromosome segregation defects and they also exhibited greater levels of genomic instability, as shown by an elevated level of centromere-positive micronuclei. In wild type HPV18 genome-containing organotypic cultures, the majority of mitotic cells reside in the suprabasal layers, in keeping with the hyperplastic morphology of the structures. However, in mutant genome-containing structures a greater proportion of mitotic cells were retained in the basal layer, which were often of undefined polarity, thus correlating with their reduced thickness. We conclude that the ability of E6 to target cellular PDZ proteins plays a critical role in maintaining mitotic stability of HPV infected cells, ensuring stable episome persistence and vegetative amplification.

The role of protein kinase A regulation of the E6 PDZ-binding domain during the differentiation-dependent life cycle of human papillomavirus type 18.

Human papillomavirus (HPV) E6 proteins of high-risk alpha types target a select group of PSD95/DLG1/ZO1 (PDZ) domain-containing proteins by using a C-terminal PDZ-binding motif (PBM), an interaction that can be negatively regulated by phosphorylation of the E6 PBM by protein kinase A (PKA). Here, we have mutated the canonical PKA recognition motif that partially overlaps with the E6 PBM in the HPV18 genome (E6153PKA) and compared the effect of this mutation on the HPVl8 life cycle in primary keratinocytes with the wild-type genome and with a second mutant genome that lacks the E6 PBM (E6ΔPDZ). Loss of PKA recognition of E6 was associated with increased growth of the genome-containing cells relative to cells carrying the wild-type genome, and upon stratification, a more hyperplastic phenotype, with an increase in the number of S-phase competent cells in the upper suprabasal layers, while the opposite was seen with the E6ΔPDZ genome. Moreover, the growth of wild-type genome-containing cells was sensitive to changes in PKA activity, and these changes were associated with increased phosphorylation of the E6 PBM. In marked contrast to E6ΔPDZ genomes, the E6153PKA mutation exhibited no deleterious effects on viral genome amplification or expression of late proteins. Our data suggest that the E6 PBM function is differentially regulated by phosphorylation in the HPV18 life cycle. We speculate that perturbation of protein kinase signaling pathways could lead to changes in E6 PBM function, which in turn could have a bearing on tumor promotion and progression.

HLA-A*02 is associated with a reduced risk and HLA-A*01 with an increased risk of developing EBV+ Hodgkin lymphoma.

Previous studies showed that the HLA class I region is associated with Epstein-Barr virus (EBV)-positive Hodgkin lymphoma (HL) and that HLA-A is the most likely candidate gene in this region. This suggests that antigenic presentation of EBV-derived peptides in the context of HLA-A is involved in the pathogenesis of EBV+ HL by precluding efficient immune responses. We genotyped exons 2 and 3, encoding the peptide-binding groove of HLA-A, for 32 single nucleotide polymorphisms in 70 patients with EBV+ HL, 31 patients with EBV- HL, and 59 control participants. HLA-A*01 was significantly overrepresented and HLA-A*02 was significantly underrepresented in patients with EBV+ HL versus controls and patients with EBV- HL. In addition, HLA-A*02 status was determined by immunohistochemistry or HLA-A*02-specific polymerase chain reaction (PCR) on 152 patients with EBV+ HL and 322 patients with EBV- HL. The percentage of HLA-A*02+ patients in the EBV+ HL group (35.5%) was significantly lower than in 6107 general control participants (53.0%) and the EBV- HL group (50.9%). Our results indicate that individuals carrying the HLA-A*02 allele have a reduced risk of developing EBV+ HL, while individuals carrying the HLA-A*01 allele have an increased risk. It is known that HLA-A*02 can present EBV-derived peptides and can evoke an effective immune response, which may explain the protective phenotype.