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.

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.

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.

Distribution and characterization of staphylococcal interspersed repeat units (SIRUs) and potential use for strain differentiation.

Variable-number tandem repeats (VNTRs) have been shown to be a powerful tool in the determination of evolutionary relationships and population genetics of bacteria. The sequencing of a number of Staphylococcus aureus genomes has allowed the identification of novel VNTR sequences in S. aureus, which are similar to those used in the study of the evolution of Mycobacterium tuberculosis clades. Seven VNTRs, termed staphylococcal interspersed repeat units (SIRUs), distributed around the genome are described, occurring in both unique and multiple sites, and varying in length from 48 to 159 bp. Variations in copy numbers were observed in all loci, within both the sequenced genomes and the UK epidemic methicillin-resistant S. aureus (EMRSA) isolates. Clonally related UK EMRSA isolates were clustered using SIRUs, which provided a greater degree of discrimination than multi-locus sequence typing, indicating that VNTRs may be a more appropriate evolutionary marker for studying transmission events and the geographical spread of S. aureus clades.

Methicillin resistant Staphylococcus aureus in the critically ill.

Methicillin resistant Staphylococcus aureus (MRSA) is endemic within many hospitals worldwide. Critically ill patients on intensive care units have increased risk factors making them especially prone to nosocomially acquired infections. This review addresses the current situation regarding the evolution of MRSA and the techniques for identifying and epidemiologically typing it. It discusses specific risk factors, the morbidity and mortality associated with critically ill patients, and possibilities for future antibiotic treatments.

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 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.

Early investigation and initiation of therapy for invasive pulmonary aspergillosis in leukaemic and bone marrow transplant patients.

Invasive fungal infections are an increasingly common problem in cancer patients and in other vulnerable groups. Invasive pulmonary aspergillosis (IPA) in the neutropenic host presents particular challenges in terms of diagnosis and therapy. Against the background of a recognized problem of invasive aspergillosis in haematology/oncology patients treated at the Christie Hospital, we undertook a prospective study in patients at risk for IPA. The aim of the study was to improve outcome by using the linked strategies of first, early diagnosis, and secondly, early aggressive therapy with a lipid-associated formulation of amphotericin B, amphotericin B colloidal dispersion ('Amphocil'). Early investigation comprised the use of high-resolution computerized tomography scanning of the thorax and fibreoptic bronchoscopy to obtain bronchoalveolar lavage specimens, processed using conventional detection and culture methods. Using this approach, the incidence of proven or probable IPA in patients with acute leukaemia was 9%. Prompt initiation of amphotericin B colloidal dispersion therapy led to a successful outcome in 11 of 13 patients, compared with a mortality of 100% in historical controls.

Detection of glycopeptide-resistant enterococci in routine diagnostic faeces specimens.

Faeces received in a diagnostic laboratory were screened for glycopeptide-resistant enterococci (GRE) on modified Lewisham medium, with and without enrichment in Enterococcosel broth. Colonization by GRE was detected in 102/838 patients (12.2%). In 74 (73%) of colonized patients GRE were detected by both methods and in 28 (27%) they were detected only after enrichment. The carriage rate in hospitalized patients was 32% (93/289) compared with 2.3% (11/425) in the community (GP patients and food-handlers). Carriage of GRE increased with age. Clostridium difficile isolation was associated with GRE colonization, odds ratio 6.76 (P<0.001). Fifty-nine percent (60/102) of the GRE had the VanA phenotype and 41% (42/102) had the VanB phenotype. In the community VanA predominated (91%), whereas 64% (57/89) of the isolates from hospitalised patients were of the VanB phenotype.

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.

Endotoxin in blood and tissue in the sudden infant death syndrome.

Although the explanation for sudden infant death syndrome (SIDS) remains unknown, an increasing body of evidence now exists to suggest a possible role for bacterial toxins in the aetiology, and a number of investigators have considered that endotoxaemia could explain some of the associated features. Following the development of an animal model which confirmed that endotoxaemia could be detected after death, we studied endotoxin levels in blood and tissue samples taken at autopsy from SIDS infants, child controls and adult controls. There were significant correlations between endotoxin levels in blood and the various organs sampled particularly in SIDS cases and child controls, and blood endotoxin levels in SIDS cases were higher in those infants where there was histological evidence of mild to moderate inflammation. However, overall no significant differences were found between endotoxin levels in blood or tissue in the three study groups. Further studies into possible actions or interactions of endotoxin in SIDS are required.

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.

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.

Improved recognition of MRSA case clusters by the application of molecular subtyping using pulsed-field gel electrophoresis.

Methicillin-resistant Staphylococcus aureus (MRSA) is increasingly common in hospital and community populations, making the recognition of true nosocomial outbreaks more difficult. We have used pulsed-field gel electrophoresis (PFGE) with Sma I digestion to analyse retrospectively two perceived outbreaks of epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA 15) colonization. The first cluster of cases in patients and staff on a general ward (ward D) revealed three different antibiograms based on differences in ciprofloxacin and rifampicin sensitivities. All isolates typed using PFGE, which was more discriminatory than phage-typing. One PFGE banding profile labelled type 5 was predominant, but 12 isolates proved to be subtypes of type 5 and two were PFGE type 11. Four staff members carried a strain not found in patients, three carried strains found in patients and transient carriage was highlighted as a problem when screening staff. PFGE enhanced the epidemiological data and proved that the cases on this ward did not comprise one large outbreak but numerous sporadic cases and smaller clusters. In contrast, isolates from a second cluster of cases which occurred on ward F were indistinguishable using antibiograms, phage-typing and PFGE, confirming this was more likely to be a true outbreak of colonization. We conclude that PFGE usefully augments epidemiological information and allows more logical infection control decisions to be made, with better utilization of scarce resources.

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.