Use of an engineered honey to eradicate preformed biofilms of important wound pathogens: an in vitro study.

OBJECTIVE: We previously reported on the ability of SurgihoneyRO (SHRO), an engineered honey, to prevent biofilm formation in vitro, but data were lacking regarding the activity against preformed biofilms. This study aims to assess whether SHRO has any antibacterial activity against mature, preformed biofilms and whether there is any evidence to support the observed clinical effectiveness when SHRO has been used anecdotally on acute and chronic wounds where biofilm is most likely present. METHOD: We tested the in vitro antibacterial activity of SHRO against the mature biofilms of 16 clinically relevant wound pathogens, in terms of impacts on biofilm seeding and biofilm biomass. The honey was serially double diluted from 1:3 down to 1:6144, and the lowest dilution achieving a statistically significant reduction in biomass of ≥50%, compared with untreated controls, was recorded. RESULTS: All 16 bacterial isolates were susceptible to SHRO, with reduced biofilm seeding observed for all, and percentage reductions ranging from 58% (ACI_C59) to 94.3% (MDR_B) for the strongest concentration of honey (1:3). Furthermore at this concentration, biofilm seeding of the test biofilm was reduced by 80-94.3% (when compared with the positive control) for 12/16 isolates. We additionally demonstrated that SHRO has antibiofilm impacts, with the 24 hour exposure resulting in disruption of the biofilm, reduced seeding and reduced biomass. CONCLUSION: SHRO is effective at reducing seeding of preformed biofilms of clinically important wound pathogens in vitro, and also has antibiofilm activity. This supports the anecdotal clinical data for antibiofilm efficacy, and supports the use of SHRO as a promising topical wound care agent.

In vitro activity of an engineered honey, medical-grade honeys, and antimicrobial wound dressings against biofilm-producing clinical bacterial isolates.

OBJECTIVE: Honey is recognised to be a good topical wound care agent owing to a broad-spectrum of antimicrobial activity combined with healing properties. Surgihoney RO (SH1) is a product based on honey that is engineered to produce enhanced reactive oxygen species (ROS) and has been reported to be highly antimicrobial. The objective was to investigate the ability of the engineered honey and its comparators to prevent biofilm formation in vitro. METHOD: We tested the ability of three medical-grade honeys SH1, Activon manuka honey (MH) and Medihoney manuka honey (Med), alongside five antimicrobial dressings (AMDs) to prevent the formation of biofilms by 16 isolates. Honeys were serially double diluted from 1:3 down to 1:6144 and the lowest dilution achieving a statistically significant reduction in biomass of at least 50%, compared with untreated controls, was recorded. RESULTS: Although all the honeys were antibacterial and were able to prevent the formation of biofilms, SH1 was the most potent, with efficacy at lower dilutions than the medical honeys for five isolates, and equivalent dilutions for a further six. Additionally, SH1 was superior in antibacterial potency to three commercially available AMDs that contain honey. CONCLUSION: SH1 is effective at preventing bioflms from forming and is superior to medical honeys and AMDs in in vitro tests. DECLARATION OF INTEREST: Surgihoney RO was provided free of charge for testing by Matoke Holdings, UK and the hospital pharmacy provided the other honeys and dressings. This paper presents independent research funded by the National Institute for Health Research (NIHR). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

Whole genome sequencing of toxigenic Clostridium difficile in asymptomatic carriers: insights into possible role in transmission.

BACKGROUND: Estimates of the prevalence of asymptomatically carried Clostridium difficile in elderly patients in long-term care range from 0% to 51%. Asymptomatic carriage is possibly a risk factor for the development of infection, and there is ongoing debate surrounding the role of asymptomatic carriage in transmission. AIM: To investigate the prevalence of asymptomatic carriage amongst patients residing in intermediate care (bedded) facilities (ICBFs), and to investigate whether asymptomatically carried C. difficile strains contribute to nosocomial C. difficile infection (CDI). METHODS: Stools were collected from eligible asymptomatic patients in ICBFs, and a subset was also processed from symptomatic patients accessing primary or secondary care outside of ICBFs. All samples were cultured for C. difficile, and resulting colonies were processed through whole genome sequencing. FINDINGS: In total, 151 asymptomatic patients were sampled, 22 of which were positive for C. difficile through stool culture, representing a carriage rate of 14.6%. Sequencing of these isolates, alongside 14 C. difficile polymerase chain reaction and culture-positive isolates from symptomatic individuals, revealed that all asymptomatic patients were carrying toxigenic C. difficile, and these strains were genetically similar to those from symptomatic patients. CONCLUSION: This small study of asymptomatic carriage revealed a rectal asymptomatic carriage rate of 14.6% in patients nursed in ICBFs, and a high level of genetic similarity of these strains to those recovered from symptomatic patients. As such, asymptomatic carriers may be important for the transmission of symptomatic CDI, although it is acknowledged that this study was small, and many other factors govern whether C. difficile is carried asymptomatically or causes symptoms.

Comparative analysis of antimicrobial susceptibility among organisms from France, Germany, Italy, Spain and the UK as part of the tigecycline evaluation and surveillance trial.

As part of the tigecycline evaluation and surveillance trial (TEST), bacterial isolates were collected from 39 centres in France, Germany, Italy, Spain and the UK between January 2004 and August 2006. Antimicrobial susceptibilities were determined according to CLSI guidelines. Italy had the highest rate of methicillin-resistant Staphylococcus aureus (36.4%), and was the only country to report vancomycin-resistant Enterococcus faecalis (8.6%). Tigecycline was the only agent to which all Gram-positive isolates were susceptible. For many of the Gram-negative organisms collected, antimicrobial susceptibilities were lowest among isolates from Italy and highest among isolates from Spain. The notable exception was Acinetobacter baumannii, where the poorest susceptibility profile was among isolates from Spain. For A. baumannii, MIC(90)s of imipenem varied from 1 mg/L for isolates in France and Germany to > or =32 mg/L for isolates from Italy and Spain. Tigecycline was the only agent to maintain an MIC(90) of < or =1 mg/L against isolates from all five countries. The in-vitro activity of tigecycline against both Gram-positive and Gram-negative isolates may make it valuable in the treatment of hospital infections, including those caused by otherwise antimicrobial-resistant organisms.

Rapid recontamination with MRSA of the environment of an intensive care unit after decontamination with hydrogen peroxide vapour.

Meticillin-resistant Staphylococcus aureus (MRSA) persists in the hospital environment and conventional cleaning procedures do not necessarily eliminate contamination. A prospective study was conducted on an intensive care unit to establish the level of environmental contamination with MRSA, assess the effectiveness of hydrogen peroxide vapour (HPV) decontamination and determine the rate of environmental recontamination. MRSA was isolated from 11.2% of environmental sites in the three months preceding the use of HPV and epidemiological typing revealed that the types circulating within the environment were similar to those colonising patients. After patient discharge and terminal cleaning using conventional methods, MRSA was isolated from five sites (17.2%). After HPV decontamination but before the readmission of patients, MRSA was not isolated from the environment. Twenty-four hours after readmitting patients, including two colonized with MRSA, the organism was isolated from five sites. The strains were indistinguishable from a strain with which a patient was colonized but were not all confined to the immediate vicinity of the colonized patient. In the eight weeks after the use of HPV, the environment was sampled on a weekly basis and MRSA was isolated from 16.3% sites. Hydrogen peroxide vapour is effective in eliminating bacteria from the environment but the rapid rate of recontamination suggests that it is not an effective means of maintaining low levels of environmental contamination in an open-plan intensive care unit.

Viridans streptococcal bacteraemia in patients with haematological and solid malignancies.

Thirty-three episodes of septicaemia caused by viridans streptococci are reported in 32 adults under treatment for malignant diseases. The underlying diseases were acute leukaemia (17), lymphoma (4), myeloma (1), small cell carcinoma of the bronchus (6), carcinoma of the breast (2) and carcinoma of the stomach (2). Important predisposing factors included severe neutropenia and oral mucositis due to intensive chemotherapeutic regimens. There was a poor response to standard empirical antibiotics and a mortality of 12%. A role for prophylactic penicillin in high risk groups is suggested.

Screening for cytomegalovirus (CMV) infection in allogeneic bone marrow transplantation using a quantitative whole blood polymerase chain reaction (PCR) method: analysis of potential risk factors for CMV infection.

Potential risk factors for CMV infection and the use of quantitative CMV PCR screening to guide pre-emptive anti-CMV therapy were reviewed retrospectively in 32 allogeneic bone marrow transplant patients accrued over a 2-year period. Significant CMV PCR positivity (an indicator of CMV infection) developed in 34% of patients. When analysed by recipient CMV IgG serostatus, 69% of seropositive recipients developed significant CMV PCR positivity while none of the seronegative recipients did so (P = 0.00007). Considering only the seropositive recipients, 100% of those who received the low intensity campath-1H/fludarabine/melphalan 'mini-allograft' conditioning regimen developed significant CMV PCR positivity, while only 44% of those who had received cyclophosphamide/TBI did so (P = 0.0337). The mean time to first episode of significant CMV PCR positivity for those who had received campath/fludarabine/melphalan was 25 days while for those who had received cyclophosphamide/TBI, this was 66 days (P = 0.0372). For the first episode of significant CMV PCR positivity, the mean index and peak CMV PCR counts for those who had received campath/fludarabine/melphalan were 4.54 and 5.22 log copies/ml respectively, while for cyclophosphamide/TBI, the corresponding figures were 3.85 and 4.12 log copies/ml respectively (P = 0.2986 and P = 0.0472 for index and peak values). 85% of those who had significant CMV PCR positivity with the campath/fludarabine/melphalan regimen developed more than one such episode, while 50% of those receiving cyclophosphamide/TBI regimen did so (P = 0.491). Significant CMV PCR positivity was associated with symptoms in a proportion of patients (pyrexia 45%, cough 18%, rise in AST 72%). No patient developed overt CMV disease. CMV PCR is useful for guiding pre-emptive anti-CMV therapy and for monitoring response.

Liposomal amphotericin (AmBisome) in the prophylaxis of fungal infections in neutropenic patients: a randomised, double-blind, placebo-controlled study.

Liposomal amphotericin (AmBisome) 2 mg/kg three times weekly was compared with placebo as prophylaxis against fungal infection in patients undergoing chemotherapy or bone marrow transplantation (BMT) for haematological malignancies. Prophylaxis began on day 1 of chemotherapy and continued until neutrophils regenerated or infection was suspected. Of 161 evaluable patients, 74 received AmBisome and 87 received placebo. Proven fungal infections developed in no patients on AmBisome and in three on placebo (3.4%) (P = NS). Suspected fungal infections requiring intervention with systemic antifungal therapy (usually amphotericin B) occurred in 31 patients on AmBisome (42%) and in 40 on placebo (46%) (P = NS). Suspected deep-seated infections developed in 21 (28.3%) and 31 (35.6%) patients, respectively (P = NS). Time to develop a suspected or proven deep-seated infection showed a trend in favour of AmBisome (P = 0.11). Fifty patients had fungal colonisation (48 with Candida spp, two with Aspergillus spp) of at least one body site during prophylaxis; 15 patients while receiving AmBisome (20%) and 35 while on placebo (40%) (P < 0.01). Time to colonisation was significantly delayed in the group receiving AmBisome (P < 0.05). Treatment-related toxicity was modest and no additional toxicity was observed in patients receiving AmBisome. AmBisome 2 mg/kg three times weekly is safe and reduces fungal colonisation in patients receiving intensive chemotherapy or BMT. However, despite encouraging trends, prophylactic AmBisome did not lead to a significant reduction in fungal infection or in requirement for systemic antifungal therapy.

Neomycin blood agar as a selective medium for vancomycin resistant Enterococcus faecium.

Neomycin blood agar is commonly used as a selective medium for the isolation of vancomycin resistant enterococci from faeces; however, not all isolates are recovered using this medium, perhaps because the neomycin concentrations are too high. To test this hypothesis, the neomycin minimum inhibitory concentration (MIC) was determined for 27 vancomycin resistant Enterococcus faecium isolates, 14 from patients with leukaemia and 13 from patients on the renal unit. A further eight isolates that had been recovered from the faeces of patients on the renal unit on neomycin agar were also studied. Eleven of the 14 isolates from the patients with leukaemia showed equal recovery on neomycin agar and blood agar and had MICs > 64 mg/l. In three other isolates there was a 4log10 reduction in recovery on neomycin agar and the MIC was 8 mg/l. Two of the non-selected isolates from the renal unit were recovered equally on the two media, the other 11 isolates showed a 4-5 log10 reduction in recovery. All eight faecal isolates recovered from patients on the renal unit on neomycin agar were highly resistant to neomycin (MIC > 64 mg/l). Comparative studies of screening media are urgently needed as vancomycin resistant enterococci become more prevalent nosocomial pathogens.

Antimicrobial activity of cytotoxic drugs may influence isolation of bacteria and fungi from blood cultures.

The potential antimicrobial activity of cyclophosphamide, vincristine, and adriamycin against Gram positive and negative bacteria and Candida albicans was examined. The time taken for different microbial inocula to turn a simulated blood culture positive in the presence of different concentrations of these drugs was measured. Doxorubicin retarded the growth of Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus sanguis in a concentration dependent manner. Cyclophosphamide and vincristine showed minimal antimicrobial activity. Escherichia coli and Pseudomonas aeruginosa were unaffected by any of the drugs. An inoculum dependent effect was seen with some combinations of microbial inocula and cytotoxic drug concentrations.

The use of continuous monitoring blood culture systems in the diagnosis of catheter related sepsis.

AIM: To assess whether the information provided by automated continuous monitoring blood culture systems could aid in the diagnosis of catheter related sepsis. METHODS: Serial dilutions of a strain of coagulase negative staphylococcus were inoculated into the BacT/Alert blood culture system. Blood culture results for seven patients with possible catheter related sepsis from coagulase negative staphylococci were reviewed. RESULTS: Time to positivity and length of lag period were strongly related to the concentration of bacteria inoculated (average decrease of 1.5 hours to positivity for each 10-fold increase in concentration). Time to positivity and length of lag period were significantly shorter for central line blood cultures than for those taken from peripheral sites. CONCLUSIONS: Using data already measured by continuous monitoring blood culture systems may provide a simple alternative to quantitative blood cultures for the diagnosis of catheter related sepsis.

Rapid identification of candida species by TaqMan PCR.

AIM: To develop and evaluate a TaqMan(TM) polymerase chain reaction (PCR) for the rapid identification and speciation of candida species. METHODS: Species specific primer and probe sets were designed for the identification of Candida albicans, C. parapsilosis, C. tropicalis, C. krusei, C. kefyr, and C. glabrata from clinical isolates in a 5' exonuclease (TaqMan(TM)) assay. The probes were labelled with three fluorescent dyes to enable differentiation between species when three primer and probe sets were combined in two multiplexes. The specificity of these assays was evaluated against a range of National Collection of Pathogenic Fungi strains, clinical isolates of yeast, bacterial and viral pathogens. RESULTS: The primer and probe sets have been shown to be 100% specific for their respective species; there was no crossreaction between any set and human DNA, or extracts from other candida species, fungal, bacterial, or viral pathogens tested. Extracts from two clinical isolates, originally identified as C albicans on the basis of germ tube formation, were not amplified by any of the primer and probe sets. These isolates have been putatively re-identified as C dubliniensis after sequencing of the variable internal transcribed spacer region ITS2 and lack of growth at 45 degrees C. CONCLUSION: This TaqMan assay provides a rapid alternative to conventional culture based techniques for the identification and speciation of the most frequently isolated candida species. The simple extraction method followed by TaqMan PCR can identify the six species mentioned in four hours.

Screening for bacterial vaginosis: a novel application of artificial nose technology.

The AromaScan system was used to analyse vaginal swabs from 68 women attending a genitourinary clinic. Using clinical criteria, subjects were assessed for bacterial vaginosis. After training the AromaScan system to recognise patterns generated from four patients with and four patients without bacterial vaginosis, 16 of the 17 (94%) remaining subjects were correctly identified as having the condition. The positive predictive value of the test was 61.5%. These results indicate that the AromaScan technology may be of value as a screening test for bacterial vaginosis.

Sleeping position and upper airways bacterial flora: relevance to cot death.

The hypothesis that the prone sleeping position is associated with accumulation of upper airways secretions and increased bacterial growth was investigated in adults. Ten subjects with upper respiratory tract infection lay prone for one hour and then supine for one hour. Nasal swabs after the prone period yielded higher bacterial counts than swabs obtained after the supine period. This result could be relevant to sudden infant death syndrome (SIDS), as infants who sleep in the prone position are at increased risk of SIDS and one theory is that death is caused by toxins produced by bacterial overgrowth in the upper respiratory tract following a viral infection.

Possibility of separating toxins from bacteria associated with sudden infant death syndrome using anion exchange chromatography.

AIMS: To develop techniques for the characterisation of toxins elaborated by a strain of Escherichia coli associated with sudden infant death syndrome (SIDS). METHODS: E coli SIDS 04, isolated from the nasopharynx of a case of SIDS, was studied. Cell-free toxin preparations were standardised, their protein measured, and analytical separation of proteins achieved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Acetone precipitation of proteins was required prior to Coomassie blue staining of bands. Preparative separation was achieved on an anion exchange column using a programmed concentration gradient of NaCl in TRIS buffer. Fractions were tested individually or pooled for presence of lethal toxin including endotoxin. Lethal toxin was detected with the chick embryo test system. Endotoxin was measured using a chromogenic modification of the Limulus amoebocyte assay. RESULTS: Twenty one peaks were detected by chromatography. Ten individual, or pooled, fractions were assayed for endotoxin which ranged from 27-33 pg/ml. Much greater variation was found when the same fractions were assayed in chick embryos. E coli fractions varied considerably in lethal toxicity, from 0/10 to 10/10 chick embryos killed/tested. Certain E coli fractions tested individually (lethality four out of 10 to eight out of 10) proved more lethal (10 out of 10) if pooled prior to testing. CONCLUSIONS: In E coli infection associated with SIDS relatively low concentrations of extracellular protein are lethally toxigenic for the chick embryo model of SIDS. These proteins can be separated analytically by SDS-PAGE and preparatively by anion exchange chromatography. Toxicity of individual fractions is not correlated with endotoxin concentrations in samples tested.

Bacterial toxins: a possible cause of cot death.

AIM: To test the hypothesis that sudden infant death syndrome (SIDS) may be caused by toxins of commonly occurring bacteria in infants lacking developed immunity. METHODS: Nasopharyngeal microbial isolates from 22 pairs of SIDS cases and healthy infants matched for age (by month), sex, and sampling time (by month) were compared for lethal toxigenicity. Crude toxin preparations were made from isolates cultured on dialysis membrane overlaid on agar, and these preparations were then tested for lethality by intravenous injection into 11 day old chick embryos. RESULTS: Fifteen (68%) of the SIDS cases were each found to have at least one lethally toxigenic organism in their nasopharyngeal flora; only eight (36%) of the flora of normal infants included a lethally toxigenic species. CONCLUSION: Infants who have died of SIDS have a significantly higher (p less than 0.05) probability than matched healthy infants of having a lethally toxigenic bacterial species in their nasopharyngeal flora.

PCR-ELISA for the early diagnosis of invasive pulmonary aspergillus infection in neutropenic patients.

AIM: To evaluate a newly developed aspergillus mitochondrial gene PCR-ELISA assay for the early diagnosis of invasive pulmonary aspergillosis (IPA) in neutropenic patients. METHODS: The aspergillus mitochondrial gene was chosen for the amplification target for use with a solution hybridisation assay with colorimetric end stage detection in microtitre plate format (PCR-ELISA). The study group comprised neutropenic patients undergoing febrile episodes not responding to standard antibacterial antibiotics. Patients underwent computed tomography and bronchoscopy. Bronchoalveolar lavage (BAL) fluids were examined by culture and PCR. RESULTS: The aspergillus mitochondrial gene PCR-ELISA was both sensitive (100%) and specific (100%) for IPA in neutropenic patients. All 12 patients with definite or probable IPA had PCR positive BAL fluids. None of the patients with undiagnosed or confirmed infections of other aetiologies were mitochondrial PCR positive. Speciation based upon amplicon size difference was possible. CONCLUSIONS: Aspergillus mitochondrial DNA PCR-ELISA on BAL fluid is useful in the early diagnosis of IPA in neutropenic patients alone or, potentially, as an indication for thoracic computed tomography.

Identification of viridans streptococci associated with bacteraemia in neutropenic cancer patients.

Twenty-three viridans streptococcal isolates from pyrexial neutropenic patients with various malignant diseases were studied in a comprehensive identification scheme. Fourteen isolates were identified as Streptococcus oralis, five as S. mitis and two as S. salivarius but the remaining two could not be identified reliably. The virulence mechanisms associated with the ability of these species to survive and grow in vivo require further investigation but may involve the production of specific glycosidase and proteolytic enzyme activities.

Subtyping of methicillin-resistant Staphylococcus aureus isolates from the North-West of England: a comparison of standardised pulsed-field gel electrophoresis with bacteriophage typing including an inter-laboratory reproducibility study.

Bacteriophage typing is currently the recognised methodology for the typing of methicillin-resistant Staphylococcus aureus (MRSA) in the UK. Bacteriophage typing is less discriminatory and does not type all isolates compared with some molecular methods for typing MRSA. Chromosomal genotyping by pulsed-field gel electrophoresis (PFGE) is increasingly recognised as an improved method for typing MRSA, providing increased discrimination and typability. In this study the results of a comparison of bacteriophage typing and PFGE typing and subtyping are presented for a large collection of isolates from the North-West of England. Isolates belonging to the most frequently isolated epidemic methicillin-resistant Staphylococcus aureus (EMRSA) bacteriophage types 15 and 16 were typed by PFGE with further discrimination of common PFGE types possible into a number of subtypes. These results for a large collection of isolates demonstrate the improved typing of MRSA with PFGE. The widespread acceptance of PFGE for typing MRSA isolates has been hampered by the lack of standardised methodologies. Recently, a standardised PFGE strain typing system, known as the GenePath system has become available. The results of an inter-laboratory comparison of PFGE typing for a collection of isolates demonstrated good reproducibility with this system.

Effect of time post mortem on the concentration of endotoxin in rat organs: implications for sudden infant death syndrome (SIDS).

The aim of the study was to test the following hypotheses: (i) that endotoxin injected 40 min prior to death can be detected in rat organs post mortem and (ii) that endotoxin levels do not change with increasing time post mortem. Rats were injected with or without endotoxin in buffered saline, 40 min prior to being killed. Endotoxin levels in rat organs were assessed using a Limulus amoebocyte assay. The effect of storage time post mortem was assessed by following various storage regimes at 25 degrees C and 8 degrees C. Significant differences (P = < 0.001) in endotoxin levels of all samples tested were found between rats injected with and without endotoxin. A significant increase in detectable endotoxin was observed between 0 h and 6 h post mortem in rats injected with or without endotoxin. No difference in detectable endotoxin levels in the kidney, liver and spleen was observed from 30 h to 102 h post mortem in rats injected with or without endotoxin. In rats injected with endotoxin, detectable endotoxin levels in the heart were raised between 0 h and 6 h, 6 h and 54 h, and 30 h and 78 h. Endotoxin injected into rats 40 min prior to death can be detected post mortem. For rats injected with saline or endotoxin prior to death levels in the kidney, liver and spleen were not affected by storage at 8 degrees C for 30-102 h, after initial storage at room temperature for 6 h. Levels of endotoxin detected in the hearts of rats injected with saline were not affected by storage up to 102 h. In rats injected with endotoxin prior to death, detectable levels in the heart were significantly affected by increasing time in storage.