Laboratory evaluation of Drawtex Hydroconductive dressing with LevaFiber technology.

OBJECTIVE: The aim of this study was to assess absorption, sequestration and retention of microorganisms by Drawtex Hydroconductive dressing with LevaFiberTM technology. METHOD: The absorption over time and the ability to sequester and retain bacteria were assessed in the laboratory using an in vitro model where known amounts of fluid and microorganisms were allowed to absorb and sequester over a 24 hour period. The reduction in numbers of microorganisms following sequestration was determined using standard plate counting methods. Retention of the organisms was visualised by scanning electron microscopy. RESULTS: Drawtex was shown to absorb eight times its own weight in fluid over time and it showed a 90% reduction in bacterial numbers over a 24 hour period in sequestration experiments. Direct observation by scanning electron microscopy demonstrated bacterial retention in the dressing fibres. CONCLUSION: Drawtex is a recent addition to the formulary for absorbent dressings available in the UK. It demonstrates excellent absorption properties, while maintaining integrity. In addition, it sequesters and retains microorganisms, which will help with removal of exudate and bioburden from the wound bed to help facilitate wound healing. DECLARATION OF INTEREST: This study was funded by an educational grant from Martindale Pharma, UK. The authors have no conflicts of interest to declare with regard to the manuscript or its content.

The benefits of silver in hygiene, personal care and healthcare.

Silver has been used for centuries as an antimicrobial agent to reduce bioburden and prevent infection. Its usage diminished when antibiotics were introduced but remained one of the most popular agents for wound infections, especially in burned patients. Incorporation of silver into a range of hygiene and healthcare applications has increased, and this has raised concerns over the development of silver resistance, toxicity, methods of testing products and evidence of efficacy. The published evidence for resistance and toxicity is limited and associated with frequent and high levels of silver used. Increasing evidence of improved antimicrobial activity of nanoparticles of silver and possible dual immunomodulatory effects are exciting. This may lead to further product development as potential alternative preservatives as some currently available preservatives have an increasing incidence of allergic reactions. Acknowledging the role of the carrier is important, and as silver is active when in solution, opens a window of opportunity in personal hygiene area. This is important in an age when multiple antibiotic-resistant bacteria are becoming prevalent.

Vapour-phase activities of essential oils against antibiotic sensitive and resistant bacteria including MRSA.

AIMS: To determine whether essential oil (EO) vapours could reduce surface and airborne levels of bacteria including methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: The antibacterial activity of geranium and lemongrass EO individually and blended were evaluated over a range of concentrations by direct contact and vapour diffusion. The EO were tested in vitro against a selection of antibiotic-sensitive and -resistant bacteria, including MRSA, vancomycin-resistant Enterococci (VRE), Acinetobacter baumanii and Clostridium difficile. An EO blend containing lemongrass and geranium was used to formulate BioScent that was dispersed into the environment using the ST Pro machine. The effects were variable depending on the methods used. In a sealed box environment, MRSA growth on seeded plates was reduced by 38% after 20 h exposure to BioScent vapour. In an office environment, the ST Pro machine dispersing BioScent effected an 89% reduction of airborne bacteria in 15 h, when operated at a constant output of 100%. CONCLUSIONS: EO vapours inhibited growth of antibiotic-sensitive and -resistant bacteria in vitro and reduced surface and airborne levels of bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that EO vapours, particularly Bioscent, could be used as a method of air disinfection.

Prevalence of toxin producing strains of Staphylococcus aureus in a pediatric burns unit.

The aims of the study were to determine the sites in a pediatric burns unit that were contaminated with Staphylococcus aureus. Samples from the environment in bedrooms and the common room were taken monthly for 6 months using blood agar for total counts and Baird-Parker agar for S. aureus. The air was sampled using an air-sampling device and settle plates. Hard and soft surfaces including bed, blanket, sofa, chair, taps, bathtub, soft toys, locker and cupboard in the same rooms were sampled using contact plates. Swabs were taken from staff monthly for 3 months. S. aureus isolates were tested for production of enterotoxins A-D and toxic shock syndrome toxin-1 using a reverse passive latex agglutination test. The results showed that S. aureus was recovered more frequently using settle plates than using the air sampler. All surfaces sampled were contaminated with S. aureus and contamination was greatest in frequently occupied rooms. A variety of toxin producing isolates were found with enterotoxin C isolates, either alone or in combination with TSST-1 (toxic shock syndrome toxin-1) dominant. The staff were transiently colonised with S. aureus strains with a different toxin production pattern. The results show that airborne transmission may be a route for infection by S. aureus and is responsible for contaminating the environment.

Antimicrobial and barrier effects of silver against methicillin-resistant Staphylococcus aureus.

OBJECTIVE: Two strains of methicillin-resistant Staphylococcus aureus (MRSA), termed epidemic strains (EMRSA-15 and EMRSA-16), were used to evaluate the antimicrobial and barrier effect of four silver dressings (two silver donating and two non-silver-donating) available in the UK at the time of the study. METHOD: The moist surface of a blood agar plate was covered with 10(6) colony-forming units of the respective strain of MRSA, and dressings were applied to the surface and incubated at 37 degrees C for different time periods and the upper and lower surfaces subcultured for residual growth. RESULTS: The nanocrystalline dressings (silver donating) were effective as a barrier from one hour until the study end (72 hours): no penetration of EMRSA-15 and EMRSA-16 through the dressing occurred. Moreover, the nanocrystalline dressings showed some antimicrobial activity at one hour in the areas underneath and surrounding the dressing until the study end. The remaining two dressings had no barrier effect and only demonstrated limited antimicrobial activity after 24 hours. CONCLUSION: This in vitro study suggests that the nanocrystalline dressings are more effective than other silver dressings in terms of providing a barrier function and antimicrobial activity against EMRSA-15 and EMRSA-16.

Optimisation of intact cell MALDI method for fingerprinting of methicillin-resistant Staphylococcus aureus.

The use of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry on intact cell microorganisms, Intact Cell MALDI (ICM), has been shown by numerous workers to yield effective species level identification. Early work highlighted the significant effect that variation in culture media, incubation conditions and length of incubation had on the spectra produced. Therefore, in order to achieve reliable and reproducible species level identification and sub-typing of microorganisms from ICM fingerprints, it has been essential to develop standardised methods. For methicillin-resistant Staphylococcus aureus (MRSA), a major nosocomial pathogen, we have developed such a standardised method. In this paper we present the experimental parameters, namely, the incubation period, the number of passages required from lyophilised or stored isolates, the method of deposition of the bacterial cells, the concentration of matrix solution, the drying time of bacterial cells prior to the addition of the matrix solution, the time between preparation of the bacterial/matrix sample and analysis and the MALDI pulsed extraction setting, which were considered during the development of defined methods.

The effect of essential oils on methicillin-resistant Staphylococcus aureus using a dressing model.

Patchouli, tea tree, geranium, lavender essential oils and Citricidal (grapefruit seed extract) were used singly and in combination to assess their anti-bacterial activity against three strains of Staphylococcus aureus: Oxford S. aureus NCTC 6571 (Oxford strain), Epidemic methicillin-resistant S. aureus (EMRSA 15) and MRSA (untypable). The individual essential oils, extracts and combinations were impregnated into filter paper discs and placed on the surface of agar plates, pre-seeded with the appropriate strain of Staphylococcus. The effects of the vapours of the oils and oil combinations were also assessed using impregnated filter paper discs that were placed on the underside of the Petri dish lid at a distance of 8mm from the bacteria. The most inhibitory combinations of oils for each strain were used in a dressing model constructed using a four layers of dressings: the primary layer consisted of either Jelonet or TelfaClear with or without Flamazine; the second was a layer of gauze, the third a layer of Gamgee and the final layer was Crepe bandage. The oil combinations were placed in either the gauze or the Gamgee layer. This four-layered dressing was placed over the seeded agar plate, incubated for 24h at 37 degrees C and the zones of inhibition measured. All experiments were repeated on three separate occasions. No anti-bacterial effects were observed when Flamazine was smeared on the gauze in the dressing model. When Telfaclear was used as the primary layer in the dressing model compared to Jelonet, greater zones of inhibition were observed. A combination of Citricidal and geranium oil showed the greatest-anti-bacterial effects against MRSA, whilst a combination of geranium and tea tree oil was most active against the methicillin-sensitive S. aureus (Oxford strain). This study demonstrates the potential of essential oils and essential oil vapours as antibacterial agents and for use in the treatment of MRSA infection.

Detection of antibodies to Staphylococcus aureus Toxic Shock Syndrome Toxin-1 using a competitive agglutination inhibition assay.

AIMS: To develop a competitive agglutination inhibition assay (CAIA) for the detection of anti-Toxic Shock Syndrome Toxin-1 (TSST-1) antibody in serum samples using a commercially available reverse passive agglutination assay (RPLA) kit. METHODS AND RESULTS: TSST-1 toxin and sera were incubated together, so that anti-toxin IgG would complex with the toxin. Latex particles sensitized with rabbit IgG anti-TSST-1 were added to test for un-complexed toxin. The sensitivity and specificity of the CAIA assay was determined relative to positive and negative ELISA results. The sensitivity (proportion of positive ELISA sera which tested positive by CAIA) was 66% whilst the specificity (proportion of ELISA negative sera which tested negative by CAIA) was 75%. Seven sera (14%) were negative by ELISA but positive for CAIA and 12 (18.8%) were positive for ELISA but negative for CAIA, suggesting some interference with the assays. Statistical analysis showed no significant difference between the methods in terms of the numbers of individuals testing positive (chi2, P = 0.04). CONCLUSIONS: The CAIA assay allowed detection of anti-TSST-1 within 18 h and was simple to read visually. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is a useful test for individual serum samples and a preliminary investigation for medical screening of suspected toxic shock syndrome and is applicable in situations where antibody assays are not routinely used for anti-TSST-1 and also where sophisticated equipment (e.g. microtitre plate reader) is not available.

Intact cell mass spectrometry (ICMS) used to type methicillin-resistant Staphylococcus aureus: media effects and inter-laboratory reproducibility.

Intact cell mass spectrometry (ICMS) rapidly analyses the surface composition of microorganisms providing rapid, discriminatory fingerprints for identification and subtyping of important nosocomial pathogens such as methicillin resistant Staphylocccus aureus (MRSA). In this study, ICMS using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI TOF/MS) was assessed for the identification and subtyping of MRSA. An intra- and inter-laboratory reproducibility study was carried out and the effects of culture media (an important source of variation for ICMS) were also studied. Several media used for the cultural identification of MRSA were examined using a panel of well-characterised staphylococcal isolates (n=26). Six MRSA isolates were analysed over a 1-month period for intra-laboratory reproducibility on the same instrument and three different culture media. Spectra were consistent for each isolate between the four experiments on the same culture medium. Individual isolates produced different spectral profiles on different culture media. Spectra from organisms grown on Columbia blood agar contained more peaks (approximately 120) compared to Columbia agar (approximately 50) and methicillin mannitol salt agar (approximately 25). All 26 staphylococcal isolates were subjected to an inter-laboratory study on two MALDI instruments. For each isolate, the overall spectral profile was the same for each of the two instruments but the baseline threshold values was adjusted due to instrument differences in detector sensitivities. Differences between certain regions of the spectra reproducibly identified isolates belonging to the two major MRSA strains (EMRSA phage group 15 and 16). These results demonstrate ICMS with appropriate media selection is a rapid and reproducible technique for identification and discrimination of MRSA.

Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR.

A single-tube 5' nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae from clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport (ctrA), capsulation (bexA), and pneumolysin (ply) gene targets specific for N. meningitidis, H. influenzae, and S. pneumoniae, respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for N. meningitidis, H. influenzae, and S. pneumoniae were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrA primers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the ply primers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the bexA primers amplified H. influenzae types b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number (n = 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal, H. influenzae, and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The ctrA primer set used in the multiplex PCR was found to be more sensitive (P < 0.0001) than the ctrA primers that had been used for meningococcal PCR testing at that time.

Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.

A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical.

A survey into toxic shock syndrome (TSS) in UK burns units.

Toxic shock syndrome (TSS) is a rare complication of a Staphylococcus aureus infection and is primarily seen in children with small burns. The true incidence of TSS in burns patients is not known and the number of presumptive cases rarely reported. This survey was undertaken to determine if the incidence of TSS in children with burns could be related to the type of dressing used to cover the wound. A questionnaire was compiled and sent to the Senior Nurse in charge of each of the UK burns units. General information on the number of admissions, age of the patient, cause of injury and burn wound management was sought. An 81% response was obtained after two mailshots and follow up telephone calls. Seventy percent (23/33) of units which answered the survey nursed children. Of these, eight units had either not encountered TSS previously or not had a case within the past two years. These units were small, admitting a maximum of 50 patients each year. Of the units where TSS was encountered, approximately 2.5% of children admitted showed symptoms of TSS. Of the units who nursed both adults and children, seven units had seen TSS in burned adult patients which has not been reported in the literature. Of the eight units where TSS had not been recently encountered, four routinely administered prophylactic antibiotics to prevent infection whereas routine administration of antibiotics occurred in only two of the 15 units where TSS was seen. Although wound management procedures differed slightly there were many similarities. These included wound cleaning with normal saline, covering with either silver sulphadiazine (1%) or povidone iodine (10%), depending upon the infection status, and dressing with a paraffin tulle, gauze and crepe bandages. No association between the management of the burn wound and subsequent development of TSS could be established.

Toxic shock syndrome toxin-1 (TSST-1) antibody levels in burned children.

Young children with burns are at risk of developing a toxic shock-like illness during the first 2-3 days after the injury. The staphylococcal exotoxin, toxic-shock syndrome toxin-1 (TSST-1) is implicated in development of this illness. Low levels or absence of anti-TSST-1 antibodies may indicate susceptibility to this illness. Anti-TSST-1 antibody levels were measured in consecutive cases admitted to the children's burns unit. Results of antibody levels in 38 of the youngest children, aged 0.04-4.0 years are reported. At the time of admission to the unit 50% of the children had IgG antibodies to TSST-1. A higher number of young burned children had antibodies to TSST-1 than expected.

Epidemiological characterization of methicillin-resistant Staphylococcus aureus isolated in the North West of England by protein A (spa) and coagulase (coa) gene polymorphisms.

In a comparative study, isolates of methicillin-resistant Staphylococcus aureus (MRSA) with known pulsed-field gel electrophoresis (PFGE) and bacteriophage type were analysed by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) for additional discriminatory subtyping information. PFGE was previously performed using standardized, commercially available kits and pre-programmed software. Isolates were examined for coagulase (coa) and protein A (spa) gene polymorphisms following PCR amplification of the coa hypervariable and spa repeat regions. Coa gene RFLPs produced a total of 38 distinct combined patterns after digestion with HaeIII and AluI and identified the predominant epidemic (EMRSA) types 15 and 16. A unique HaeIII restriction site was identified by RFLP and sequence analysis in the coa gene for EMRSA 15 but not EMRSA 16. The spa gene PCR yielded a total of 14 different profiles ranging from 3-18 repeats with the 2 predominant EMRSA types falling into 2 distinct groups. PCR detection of coa and spa polymorphisms offer a rapid preliminary strain identification and discriminatory subtyping information for surveillance of MRSA.

Metronidazole sensitivity testing of Helicobacter pylori: the importance of media.

Successful triple-therapy treatment for Helicobacter pylori infection depends upon metronidazole (Mz) susceptibility, and many hospital laboratories routinely screen H. pylori isolates for Mz resistance using disc diffusion methods. We report the importance of culture medium when testing for metronidazole susceptibility. In this laboratory, Mz resistance in strains of H. pylori from patients in our area was found in approximately 80%. In other areas, Mz resistance is found in approximately 30%. This high rate of Mz resistance was not reflected clinically. Added haemin (X factor) and menadione in the culture medium drastically reduced zone size to Mz and also interfered with the minimal inhibitory concentration (MIC) as determined by Etest. When strains of H. pylori were re-tested on media which did not contain X factor or menadione, Mz resistance fell from 80% to 39%, a level similar to that seen in other areas.

Toxic shock syndrome in the burned patient.

Toxic shock syndrome (TSS) has gained notoriety because of its association with tampon use. However, there is an increasing awareness of the syndrome on many of the specialised burn units in hospitals through the United Kingdom. TSS primarily affects children with small-percentage burns, and it is this group of patients that normally would be expected to make an uneventful recovery. One unit, where 100-150 children are admitted per year, has seen four cases of confirmed TSS over a two-year period. There does not appear to be the same risk of TSS in adult burned patients, and this lower incidence may be the result of an increase in the production of antibodies to toxic shock toxins with increase in age.

The prevalence of Enterocytozoon bieneusi in acquired immunodeficiency syndrome (AIDS) patients from the north west of England: 1992-1995.

Microsporidia are increasingly being recognised as important enteric pathogens in patients with advanced human immunodeficiency virus (HIV) disease, i.e. acquired immunodeficiency syndrome (AIDS). The aims of this study were to investigate the frequency of detection of microsporidia associated with diarrhoea in patients with advanced HIV disease in the north west of England, and to determine the species involved and their prevalence. During the period from April 1992 to the end of December 1995, chronic diarrhoea in 88 patients in the late stage of HIV disease was investigated. Duodenal biopsies, duodenal aspirates or jejunal biopsies were received from 38 patients, and stool samples from 63 patients, as part of the routine investigation of possible causes of diarrhoea in these patients. Biopsies and aspirates were examined by thin-section electron microscopy (EM), and stool samples were examined by epi-fluorescence microscopy after staining with Calcofluor. Putative stool positives were confirmed by transmission electron microscopy. CD4-lymphocyte counts were available from all patients who provided samples. Nine out of 63 patients (14.3%) were found to be excreting microsporidial spores on stool examination. The species was confirmed as Enterocytozoon bieneusi. The mean CD4-lymphocyte count for this group was 37 x 10(6)/L (normal range 517-1677 x 10(6)/L). Three out of 38 biopsy specimens (7.9%) were also found to be infected with this microsporidian. The mean CD4-lymphocyte count for this group was 72 x 10(6)/L. Encephalitozoon intestinalis was not found in any samples examined. The prevalence of microsporidial infection in AIDS patients in the north west of England appears to be similar to that of patients in London, but less than that reported in studies from other developed countries.

The rapid identification of intact microorganisms using mass spectrometry.

Antibiotic-resistant strains of bacteria continue to emerge, increasing the need for their fast and accurate identification. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), has become a prominent technique in biological mass spectrometry. We report the application of MALDI-TOF-MS for the identification of intact Gram-negative and Gram-positive microorganisms taken directly from culture. Analysis of bacteria from a single colony is possible, allowing the screening of mixed cultures. Sample preparation is simple and the analysis automated, providing spectra within minutes. The spectra obtained allow identification of microorganisms from different genera, different species, and from different strains of the same species. The procedure provides a unique mass spectral fingerprint of the microorganism, produced from desorbed components of the cell wall. Consistent data were obtained from subcultures grown for 3-day and 6-day periods, from the same cultures 1 day later and from fresh subcultures 2 months later.

A comparison of phenotypic properties of Staphylococcus aureus isolated from burned children and other patient groups.

This study aimed to determine whether strains of Staph. aureus isolated from children on our paediatric burns unit were different from strains isolated from other patient groups. Of particular interest was the incidence of toxin production amongst the different patient groups and the potential association with toxic shock syndrome (TSS). Wound isolates of Staph. aureus were collected from three patient groups: (1) hospital inpatients, (2) community patients and (3) patients from a regional burns unit. One hundred isolates were collected from each group (n = 300). Each isolate was tested for enterotoxin and TSST-1 production, phage type, antibiogram and tryptophan dependence. The results were compared, to determine whether there were any differences between the isolates from each of these patient groups. There were some variations in antibiotic sensitivity patterns and phage type of the isolates between the different patient groups but there was no significant difference in the incidence of toxin production, which was an important observation. The 100 isolates collected from this burns unit were derived from 58 patients. The colonization patterns of the Staph. aureus showed that 12 patients were colonized by more than one isolate and that these were a mixture of toxin-positive and toxin-negative strains. The medical records were examined for evidence of TSS; there was a higher incidence of toxic episodes in the patients colonized with strains which produced TSST-1 toxin.

The effect of topical antimicrobial agents on the production of toxic shock syndrome toxin-1.

Three hundred isolates of Staphylococcus aureus from wound swabs were examined for the production of toxic shock syndrome toxin 1 (TSST-1). The isolates were collected from community patients, surgical inpatients and from patients in the Regional Burns Unit, Booth Hall Children's Hospital, Manchester. The overall incidence of toxin production was 17% and there was no significant variation between the sources of the strains. All 55 TSST-1-producing strains were grown in sublethal concentrations of five topical antimicrobial compounds and the level of toxin produced was determined and compared with the amount produced in a control broth after incubation for 24 h. The effects of sublethal concentrations of the compounds on TSST-1 production were strain dependent; some compounds tended to increase production (at least four-fold) and some tended to decrease production (at least four-fold). Some of the strains showed an increase in toxin production in the presence of chlorhexidine gluconate/cetrimide solution and silver sulphadiazine cream whereas 18%, 42% and 47% of the strains showed a decrease in toxin production in the presence of povidone iodine solution, stabilised hydrogen peroxide cream and mupirocin ointment, respectively. Preliminary results suggest that silver sulphadiazine cream induces toxin formation earlier in the growth cycle.