Protein, cell and bacterial response to atmospheric pressure plasma grafted hyaluronic acid on poly(methylmethacrylate).

Hyaluronic acid (HA) has been immobilised on poly(methyl methacrylate) (PMMA) surfaces using a novel dielectric barrier discharge (DBD) plasma process for the purposes of repelling protein, cellular and bacterial adhesion in the context of improving the performance of ophthalmic devices. Grafting was achieved by the following steps: (1) treatment of the PMMA with a DBD plasma operating at atmospheric pressure, (2) amine functionalisation of the activated polymer surface by exposure to a 3-aminopropyltrimethoxysilane (APTMS) linker molecule and (3) reaction of HA with the surface bound amine. The mechanism and effectiveness of the grafting process was verified by surface analysis. XPS data indicates that the APTMS linker molecule binds to PMMA via the Si-O chemistry and has the required pendant amine moiety. The carboxylic acid moiety on HA then binds with this -NH2 group via standard carbodiimide chemistry. ToF-SIMS confirms the presence of a coherent HA layer the microstructure of which is verified by AFM. The plasma grafted HA coating surfaces showed a pronounced decrease in protein and cellular adhesion when tested with bovine serum albumin and human corneal epithelial cells, respectively. The ability of these coatings to resist bacterial adhesion was established using Staphylococcus aureus NTC8325. Interestingly, the coatings did not repel bacterial adhesion, indicating that the mechanism of adhesion of bacterial cells is different to that for the surface interactions of mammalian cells. It is proposed that this difference is a consequence of the specific HA conformation that occurs under the conditions employed here. Hence, it is apparent that the microstructure/architecture of the HA coatings is an important factor in fabricating surfaces intended to repel proteins, mammalian and bacterial cells.

Atmospheric pressure plasma induced grafting of poly(ethylene glycol) onto silicone elastomers for controlling biological response.

This study investigates the role that surface functionalisation of silicone elastomer (SE) by atmospheric pressure plasma induced graft immobilisation of poly(ethylene glycol) methyl ether methacrylate (PEGMA) plays in the attendant biological response. SE is used in modern ophthalmic medical devices and samples of the material were initially plasma treated using a dielectric barrier discharge reactor (DBD) to introduce reactive oxygen functionalities, prior to in situ grafting of two molecular weights of PEGMA (MW 1000 Da: PEGMA(1000), MW 2000 Da: PEGMA(2000)). The variously processed surfaces were characterised by water contact angle analysis, X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry and atomic force microscopy. Lens epithelial cells were then cultured on the PEGMA grafted SE surfaces. It was found that cells on the pristine surface were not well spread and had shrunken morphology. On the DBD pre-treated surfaces, the cells were well spread. On the PEGMA(1000) surface, the cells displayed evidence of shrinkage and were on the verge of detaching. Remarkably, on the PEGMA(2000) surface, no cell adhesion was detection. Bacterial adhesion to the surfaces was studied using Staphylococcus aureus NTC8325. There was no difference in the number of bacteria adhering to any of the surfaces studied.

Screening of clinical, food, water and animal isolates of Escherichia coli for the presence of blaCTX-M extended spectrum beta-lactamase (ESBL) antibiotic resistance gene loci.

A small study was carried out in order to examine the molecular presence of bla CTX-M gene phylogenetic groups in E. coli (n=263) isolated from food (n=54), water (n=7), animal sources (n=69), using consensus bla CTX-M primers and PCR, in addition to human faecal isolates (n=69) and VTEC O157:H7 (n=64). None of the clinically significant faecal VTEC O157:H7 isolates were shown to carry blaCTX-M type phylogenetic groups, nor were such phylogenetic groups observed in any of the food, water and animal isolates. One community faecal isolate (1/69; 1.4%), dating from 1997, carried this phylogenetic group. As recent work has indicated that a significant proportion of such phylogenetic groups are carried in community isolates of E. coli with little or no hospital contact, it is important that surveillance is increased to identify potential source(s) and reservoirs of such resistance in the community. Further prospective surveillance is thus required to help elucidate the origins of such phylogenetic group in the community. The significance of this study is that the ESBL-producing E. coli associated with local hospital outbreaks is not commonly found in local food, water or animal sources. In addition, given that ESBL-producing E. coli is now a significant organism, both in hospitals and nursing homes in Northern Ireland, this report demonstrates that such organisms were present in the community, as early as 1997.

Molecular identification of airborne bacteria associated with aerial spraying of bovine slurry waste employing 16S rRNA gene PCR and gene sequencing techniques.

Polymerase chain reaction amplification of the universal 16S ribosomal RNA (rRNA) gene was performed on a collection of 38 bacterial isolates, originating from air sampled immediately adjacent to the agricultural spreading of bovine slurry. A total of 16 bacterial genera were identified including both Gram-positive and Gram-negative genera. Gram-positive organisms accounted for 34/38 (89.5%) of total bacterial numbers consisting of 12 genera and included Staphylococcus (most common genus isolated), Arthrobacter (2nd most common genus isolated), Brachybacterium, Exiguobacterium, Lactococcus, Microbacterium and Sporosarcina (next most common genera isolated) and finally, Bacillus, Brevibacterium, Frigoribacterium, Mycoplana and Pseudoclavibacter. Gram-negative organisms accounted for only 4/38 (10.5%) bacterial isolates and included the following genera, Brevundimonas, Lysobacter, Psychrobacter and Rhizobium. No gastrointestinal pathogens were detected. Although this study demonstrated a high diversity of the microorganisms present, only a few have been shown to be opportunistically pathogenic to humans and none of these organisms described have been described previously as having an inhalational route of infection and therefore we do not believe that the species of organisms identified pose a significant health and safety threat for immunocompetant individuals.

Epidemiological study of E. coli O157:H7 isolated in Northern Ireland using pulsed-field gel electrophoresis (PFGE).

In Northern Ireland over the last 7 years, there is a mean of 41.9 laboratory reports per annum of human gastrointestinal infection (range 19-54) caused by Escherichia coli O157:H7. In the preceding years 1992-1996, reports were 5.4 per annum, whereas in 1997-2000, reports increased from 30 to 54 per annum. This high level has continued on an annual basis to date. The aim of this study was therefore to retrospectively examine this period of exponential increase in reports to help ascertain the genetic relatedness of strains employing pulsed-field gel electrophoresis (PFGE), as no data on the molecular epidemiology of E. coli O157:H7 in Northern Ireland has yet been published. Clinical isolates (n=84) were PFGE typed employing XbaI digestion and resulting band profiles demonstrated the presence of 13, 9 and 16 clonal types, for 1997, 1998 and 1999, respectively. In 1998, five clonal types remained from 1997 with the introduction of 4 new clonal types, whereas in 1999, 10 new clonal types were observed, accounting for over half (58%) of the E. coli O157 isolates for that year. These data suggest that, unlike gastrointestinal infections due to thermophilic campylobacters, there was considerable genetic evolution ofPFGE clonal types of E. coli O157, through the displacement and emergence of genotypes. Further studies are now required to find the environmental reservoirs of these common clonal types of clinical E. coli O157:H7 in Northern Ireland to help define sources and routes of transmission of this infection locally.

Comparison of the effect of ciprofloxacin and Tazocin on the incidence of meticillin-resistant Staphylococcus aureus (MRSA) in an Intensive Care Unit.

Meticillin-resistant Staphylococcus aureus (MRSA) is a very significant agent of recalcitrant healthcare-associated infections. A major risk of acquiring such infections is thought to be modulated by the use of particular antimicrobial therapies. The aim of this research was to evaluate prospectively the impact of using either ciprofloxacin or Tazocin (piperacillin+tazobactam) on the incidence of MRSA in an Intensive Care Unit (ICU). The 1-year (2 x 6 months) cross-over study was carried out in a medium-sized (426 beds) teaching hospital. During the first 6-month period, ciprofloxacin was used as the first-line broad-spectrum antibiotic therapy of choice. During the second 6-month period, Tazocin was used as first-line therapy. The incidence of hospital-acquired MRSA (i.e. colonised and/or infected) and rates of compliance of the ICU healthcare workers to optimal hand hygiene practices were recorded throughout the study. The study observed no statistically significant differences (P = 0.1) between MRSA incidence rates in the ICU during the ciprofloxacin (4.4/1000 bed-days) or Tazocin (11.4/1000 bed-days) arms of the study. Interestingly, observing healthcare workers' hand hygiene practices throughout the entire study showed that healthcare workers adhered to these practices 59.2% of the time during the ciprofloxacin arm and 66.0% during the Tazocin arm. The low incidence rates within the unit demonstrated the importance of infection control in limiting the spread of MRSA despite the extensive use of antibiotics in a high-risk setting.

Environmental stress and antibiotic resistance in food-related pathogens.

This study investigated the possibility that sublethal food preservation stresses (high or low temperature and osmotic and pH stress) can lead to changes in the nature and scale of antibiotic resistance (ABR) expressed by three food-related pathogens (Escherichia coli, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus). The study found that some sublethal stresses significantly altered antibiotic resistance. Incubation at sublethal high temperature (45 degrees C) decreased ABR. Incubation under increased salt (>4.5%) or reduced pH (<5.0) conditions increased ABR. Some of the pathogens continued to express higher levels of ABR after removal of stress, suggesting that in some cases the applied sublethal stress had induced stable increases in ABR. These results indicate that increased use of bacteriostatic (sublethal), rather than bactericidal (lethal), food preservation systems may be contributing to the development and dissemination of ABR among important food-borne pathogens.

Habituation to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia) is associated with reduced susceptibility to antibiotics in human pathogens.

OBJECTIVES: To investigate the effect of sub-lethal challenge with tea tree oil (TTO) on the antibiotic susceptibility profiles of significant human pathogens and commensals. METHODS: The study compared the antibiotic susceptibility (Etest) patterns of Escherichia coli, Staphylococcus aureus/methicillin-resistant S. aureus (MRSA) and Salmonella spp. after broth culture for 72 h in the presence or absence of sub-lethal concentrations of TTO (0.25%, 0.25% and 0.1%). RESULTS: All habituated cultures (exposed to sub-lethal concentrations of TTO) displayed reduced susceptibility to a range of clinically relevant antibiotics compared with non-habituated (control) cultures. CONCLUSIONS: Although TTO may be an effective antimicrobial agent when appropriately used at bactericidal concentrations, its application at sub-lethal concentrations may contribute to the development of antibiotic resistance in human pathogens.