Convergent evolution of pain-inducing defensive venom components in spitting cobras.

Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A2 (PLA2) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense.

Difficulty in removing biofilm from dry surfaces.

Cleaning is fundamental to infection control. This report demonstrates that a Staphylococcus aureus biofilm is significantly more difficult to remove than dried planktonic bacteria. A single wiping action removed >99.9% (>3 log10) of dried planktonic bacteria, whereas only 1.4 log10 of biofilm (96.66%) was removed by 50 wiping actions with a standardized wiping process.

Transfer of dry surface biofilm in the healthcare environment: the role of healthcare workers' hands as vehicles.

BACKGROUND: Dry surface biofilms (DSBs) persist for extended periods in hospital, and may play a significant role in transmission of healthcare-associated infections. AIM: To determine whether DSBs may be transferred from hospital surfaces to healthcare workers' hands. METHOD: Twelve-day Staphylococcus aureus DSB was grown on polycarbonate and glass coupons in a CDC Biofilm Reactor®. A total of 1.8 × 106 and 8.8 × 105 bacteria grew on the polycarbonate and glass coupons respectively. Transmission was tested by lifting the coupon with forefinger and thumb of ungloved hands to a height of 30 cm, then touching horse blood agar (HBA) plates 19 sequential times. Transferred bacterial number was determined by colony-forming units. The effect of DSB wetting on biofilm transfer was tested with 5% neutral detergent treatment for 5 s. FINDINGS: Between 5.5 and 6.6% of the DSB bacteria were transferred to hands with one touch and ∼20% were then transferred to HBA with one touch, giving an overall transfer rate of 1.26% and 1.04% for polycarbonate and glass coupons, respectively. Detergent treatment had little effect on bacterial removal from coupons, but, for biofilm grown on polycarbonate, significantly increased transferral to HBA (P < 0.001) to 5.2%. Large numbers of bacteria were transferred by bare hands to multiple fomites. One-third of polycarbonate coupons transferred >1000 colonies during the first five sequential touches. Sufficient bacteria to cause infection were transmitted up to 19 times following one touch of the DSB. CONCLUSION: DSB bacteria are transferred by hands from one fomite to multiple fomites, suggesting that DSB may serve as a persistent environmental source of pathogens.

Staphylococcus aureus dry-surface biofilms are more resistant to heat treatment than traditional hydrated biofilms.

BACKGROUND: The importance of biofilms to clinical practice is being increasingly realized. Biofilm tolerance to antibiotics is well described but limited work has been conducted on the efficacy of heat disinfection and sterilization against biofilms. AIM: To test the susceptibility of planktonic, hydrated biofilm and dry-surface biofilm forms of Staphylococcus aureus, to dry-heat and wet-heat treatments. METHODS: S. aureus was grown as both hydrated biofilm and dry-surface biofilm in the CDC biofilm generator. Biofilm was subjected to a range of temperatures in a hot-air oven (dry heat), water bath or autoclave (wet heat). FINDINGS: Dry-surface biofilms remained culture positive even when treated with the harshest dry-heat condition of 100°C for 60min. Following autoclaving samples were culture negative but 62-74% of bacteria in dry-surface biofilms remained alive as demonstrated by live/dead staining and confocal microscopy. Dry-surface biofilms subjected to autoclaving at 121°C for up to 30min recovered and released planktonic cells. Recovery did not occur following autoclaving for longer or at 134°C, at least during the time-period tested. Hydrated biofilm recovered following dry-heat treatment up to 100°C for 10min but failed to recover following autoclaving despite the presence of 43-60% live cells as demonstrated by live/dead staining. CONCLUSION: S. aureus dry-surface biofilms are less susceptible to killing by dry heat and steam autoclaving than hydrated biofilms, which are less susceptible to heat treatment than planktonic suspensions.

Determination of bacterial species present in biofilm contaminating the channels of clinical endoscopes.

BACKGROUND: Outbreaks of endoscopy-related Carbapenem-resistant Enterobacteriaceae has highlighted failures in endoscope decontamination resulting in biofilm formation. Biofilms are tolerant to detergents and disinfectants. We evaluated decontaminated endoscope channels for residual bacterial contamination and biofilm presence. METHODS: 64 channels were collected from 12 gastroscopes and 11 colonoscopes. Aerobic bacteria were isolated from inside the endoscope tubing by scrapping, sonication, and aerobic plate culture. Total number of contaminating bacteria was determined by quantitative real-time PCR with 16s rRNA eubacterial universal primers. Microbial diversity was assessed using next generation DNA sequencing. Biofilm presence was visually confirmed by confocal laser scanning and scanning electron microscopy. RESULTS: 47% of channels were culture positive, with α-haemolytic Streptococci from gastroscopes and coliforms from colonoscopes the most frequently isolated species. Sphingomonas spp., Staphylococcus spp., Streptococcus spp., and Pseudomonas aeruginosa were also isolated. An average of 1.2 × 103 bacteria/cm contaminated air-water channels, 2.8 × 102 and 6.6 × 102 bacteria/cm contaminated gastroscope and colonoscope working channels, respectively. Biofilm was on all 39 channels examined and was principally composed of environmental bacteria, although all samples contained potential pathogens. CONCLUSION: Biofilm is present on many endoscope channels obtained from Australian hospitals. Any soil including biofilm can compromise disinfectant action.

Comparison of preterm and term equivalent age MRI for the evaluation of preterm brain injury.

OBJECTIVE: To compare information obtained from preterm magnetic resonance imaging (MRI; 31-34 weeks) brain scan to that done at term equivalent age. STUDY DESIGN: Prospective observational study of premature infants with evidence or suspicion of parenchymal brain injury on cranial ultrasound. Brain injury on two scans scored using a scoring system and analyzed. RESULTS: Fourteen infants with a median (range) gestation at birth of 28 (25-29) weeks and birth weight of 1254 (680-1557) grams were studied. There was a strong correlation between the brain injury scores for the two scans (Spearman ρ=0.87, P=0.001) with excellent agreement between two radiologists (interclass correlation coefficient 0.9-0.94). There was also a high level of agreement between the preterm and term MRI two scores (Intraclass correlation coefficient, 0.79 (0.53-0.94)). CONCLUSIONS: Preterm MRI is a feasible option for the assessment of preterm brain injury and analysis of data obtained from scan at preterm age is comparable to that obtained at term equivalent age.

Staphylococcus aureus dry-surface biofilms are not killed by sodium hypochlorite: implications for infection control.

BACKGROUND: Dry hospital environments are contaminated with pathogenic bacteria in biofilms, which suggests that current cleaning practices and disinfectants are failing. AIM: To test the efficacy of sodium hypochlorite solution against Staphylococcus aureus dry-surface biofilms. METHODS: The Centers for Disease Control and Prevention Biofilm Reactor was adapted to create a dry-surface biofilm, containing 1.36 × 10(7)S. aureus/coupon, by alternating cycles of growth and dehydration over 12 days. Biofilm was detected qualitatively using live/dead stain confocal laser scanning microscopy (CLSM), and quantitatively with sonicated viable plate counts and crystal violet assay. Sodium hypochlorite (1000-20,000parts per million) was applied to the dry-surface biofilm for 10min, coupons were rinsed three times, and residual biofilm viability was determined by CLSM, plate counts and prolonged culture up to 16 days. Isolates before and after exposure underwent minimum inhibitory concentration (MIC) and minimum eradication concentration (MEC) testing, and one pair underwent whole-genome sequencing. FINDINGS: Hypochlorite exposure reduced plate counts by a factor of 7 log10, and reduced biofilm biomass by a factor of 100; however, staining of residual biofilm showed that live S. aureus cells remained. On prolonged incubation, S. aureus regrew and formed biofilms. Post-exposure S. aureus isolates had MICs and MECs that were not significantly different from the parent strains. Whole-genome sequencing of one pre- and post-exposure pair found that they were virtually identical. CONCLUSIONS: Hypochlorite exposure led to a 7-log kill but the organisms regrew. No resistance mutations occurred, implying that hypochlorite resistance is an intrinsic property of S. aureus biofilms. The clinical significance of this warrants further study.

Intensive care unit environmental surfaces are contaminated by multidrug-resistant bacteria in biofilms: combined results of conventional culture, pyrosequencing, scanning electron microscopy, and confocal laser microscopy.

BACKGROUND: Hospital-associated infections cause considerable morbidity and mortality, and are expensive to treat. Organisms causing these infections can be sourced from the inanimate environment around a patient. Could the difficulty in eradicating these organisms from the environment be because they reside in dry surface biofilms? AIM: The intensive care unit (ICU) of a tertiary referral hospital was decommissioned and the opportunity to destructively sample clinical surfaces was taken in order to investigate whether multidrug-resistant organisms (MDROs) had survived the decommissioning process and whether they were present in biofilms. METHODS: The ICU had two 'terminal cleans' with 500 ppm free chlorine solution; items from bedding, surrounds, and furnishings were then sampled with cutting implements. Sections were sonicated in tryptone soya broth and inoculated on to chromogenic plates to demonstrate MDROs, which were confirmed with the Vitek2 system. Genomic DNA was extracted directly from ICU samples, and subjected to polymerase chain reaction (PCR) for femA to detect Staphylococcus aureus and the microbiome by bacterial tag-encoded FLX amplicon pyrosequencing. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were performed on environmental samples. FINDINGS: Multidrug-resistant bacteria were cultured from 52% (23/44) of samples cultured. S. aureus PCR was positive in 50%. Biofilm was demonstrated in 93% (41/44) of samples by CLSM and/or SEM. Pyrosequencing demonstrated that the biofilms were polymicrobial and contained species that had multidrug-resistant strains. CONCLUSION: Dry surface biofilms containing MDROs are found on ICU surfaces despite terminal cleaning with chlorine solution. How these arise and how they might be removed requires further study.

Early MRI in term infants with perinatal hypoxic-ischaemic brain injury: interobserver agreement and MRI predictors of outcome at 2 years.

AIM: To compare diffusion-weighted imaging (DWI) and non-DWI magnetic resonance imaging (MRI), proton MR spectroscopy (1H-MRS), and clinical biomarkers for prediction of 2 year developmental outcome in term infants with perinatal hypoxic-ischaemic encephalopathy (HIE). MATERIALS AND METHODS: Nineteen infants ≥36 weeks gestation with HIE were recruited and MRI performed day 3-7 (mean = 5). MRI was scored independently by three radiologists using a standardized scoring system. Lactate-to-N-acetylaspartate ratio (Lac:NAA) in the lentiform nucleus was calculated. Developmental assessment was performed at 2 years using the Bayley Scales of Infant and Toddler Development (BSID-III). Interobserver agreement about abnormality in 10 brain regions was measured. Univariate analysis was performed to determine variables associated with adverse outcome (i.e., death or Bayley score for any domain <70). RESULTS: Good interobserver agreement (kappa = 0.61-0.69) on scores for DWI was obtained for the cortex, putamen, and brainstem, but not for any region on non-DWI. A significant association was found between outcome and Lac:NAA (p < 0.003) and DWI scores for lentiform nucleus, thalamus, cortex, posterior limb of the internal capsule (PLIC), and paracentral white matter (p = 0.001-0.013), but for non-DWI score only in the vermis or brainstem. A combination of Lac:NAA ≥0.25 or DWI/apparent diffusion coefficient (ADC) signal abnormality in the PLIC had 100% specificity and sensitivity for poor outcome. CONCLUSION: Interobserver agreement for non-DWI performed during the first week is poor. Agreement by three radiologists about the presence of abnormal signal within the PLIC on ADC/DWI images or elevation of Lac:NAA above 0.25 improved sensitivity without reducing the prognostic specificity of MRS in the 19 patients, but this requires validation in a larger group of infants with HIE who have been treated with hypothermia.

Spontaneous oesophageal perforations in a competitive swimmer.

Oesophageal perforation is a rare but potentially life-threatening condition. We report a case of a young male athlete who presented to our unit with two separate areas of spontaneous oesophageal perforation.

Investigation of inter-individual variability of the one-carbon folate pathway: a bioinformatic and genetic review.

Genetic polymorphisms in the one-carbon folate pathway have been widely studied in association with a number of conditions. Most of the research has focused on the 677C>T polymorphism in the coding region of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene. However, there are a total of 25 genes in this pathway coding for enzymes, transporters and receptors, which can be investigated using 267 tagging single nucleotide polymorphisms (SNPs); using SNP database (dbSNP), 38 non-synonymous SNPs with a minor allele frequency of >5% are present in these genes. Most of these variants have not been investigated in relation to disease or drug response phenotypes. In addition, their functional consequences are largely unknown. Prediction of the functional effect using six publicly available programs (PolyPhen, SIFT BLink, PMut, SNPs3D, I-Mutant2.0 and LS-SNP) was limited to functionally well-characterized SNPs such as MTHFR c.677C>T and c.1298A>C ranking low. Epigenetic modifications may also be important with some of these genes. In summary, to date, investigation of the one-carbon folate pathway genes has been limited. Future studies should aim for a more comprehensive assessment of this pathway, while further research is also required in determining the functional effects of these genetic variants.

Ovarian cancer in the proteomics era.

Ovarian cancer presents a diagnostic challenge because of its subtle clinical presentation and elusive cell of origin. Two new technologies of proteomics have advanced the dissection of the underlying molecular signaling events and the proteomic characterization of ovarian cancer: mass spectrometry and protein array analysis. Mass spectrometry can provide a snapshot of a proteome in time and space, with sensitivity and resolution that may allow identification of the elusive "needle in the haystack" heralding ovarian cancer. Proteomic profiling of tumor tissue samples can survey molecular targets during treatment and quantify changes using reverse phase protein arrays generated from tumor samples captured by microdissection, lysed and spotted in serial dilutions for high-throughput analysis. This approach can be applied to identify the optimal biological dose of a targeted agent and to validate target to outcome link. The evolution of proteomic technologies has the capacity to advance rapidly our understanding of ovarian cancer at a molecular level and thus elucidate new directions for the treatment of this disease.

Differentiation of tumour-stage mycosis fungoides, psoriasis vulgaris and normal controls in a pilot study using serum proteomic analysis.

BACKGROUND: Serum proteomic analysis is an analytical technique utilizing high-throughput mass spectrometry (MS) in order to assay thousands of serum proteins simultaneously. The resultant 'proteomic signature' has been used to differentiate benign and malignant diseases, enable disease prognosis, and monitor response to therapy. OBJECTIVES: This pilot study was designed to determine if serum protein patterns could be used to distinguish patients with tumour-stage mycosis fungoides (MF) from patients with a benign inflammatory skin condition (psoriasis) and/or subjects with healthy skin. METHODS: Serum was analysed from 45 patients with tumour-stage MF, 56 patients with psoriasis, and 47 controls using two MS platforms of differing resolution. An artificial intelligence-based classification model was constructed to predict the presence of the disease state based on the serum proteomic signature. RESULTS: Based on data from an independent testing set (14-16 subjects in each group), MF was distinguished from psoriasis with 78.6% (or 78.6%) sensitivity and 86.7% (or 93.8%) specificity, while sera from patients with psoriasis were distinguished from those of nonaffected controls with 86.7% (or 93.8%) sensitivity and 75.0% (or 76.9%) specificity (depending on the MS platform used). MF was distinguished from unaffected controls with 61.5% (or 71.4%) sensitivity and 91.7% (or 92.9%) specificity. In addition, a secondary survival analysis using 11 MS peaks identified significant survival differences between two MF groups (all P-values <0.05). CONCLUSIONS: Serum proteomics should be further investigated for its potential to identify patients with neoplastic skin disease and its ability to determine disease prognosis.

Relaparotomy: a five-year review of indications and outcome.

Complications from abdominal surgery may necessitate reoperation and can be associated with significant morbidity and mortality. This review aims to analyse the incidence and outcome of relaparotomy for various indications. In a retrospective review of case notes of patients who had undergone one or more relaparotomies during the same hospitalisation between 1996 and 2000, 55 patients required relaparotomy. Indications included bleeding, infection, anastomotic leakage, wound dehiscence, necrotising pancreatitis, bowel necrosis, bowel obstruction and miscellaneous indications. Relaparotomy for dehiscence and obstruction carried minimal risk; for bleeding and infection entailed moderate risks; and for anastomotic leak had the highest mortality rate. The mortality rate increased in older age groups, multiple system and organ failure and multiple relaparotomies. The overall mortality rate was 38%. Twenty-nine per cent of patients had MRSA infection contributing to sepsis and multiple system and organ failure. Reintervention had brought to evidence technical errors, which could be corrected, and resulted in patient salvage in some cases. The mortality rate of relaparotomy has remained unchanged compared with data published previously, despite improvements in surgical techniques and critical care. Timely relaparotomy is valuable in the identification and treatment of complications following abdominal surgery.

Recognition of multiple classes of hepatitis C antibodies increases detection sensitivity in oral fluid.

Paired serum-oral fluid samples from 127 hepatitis C virus (HCV)-positive and 31 HCV-negative patients were tested for the presence of anti-HCV using the Ortho HCV 3.0 ELISA. Using the immunoglobulin G (IgG)-specific detection antibody provided with the HCV 3.0 ELISA we attained 100% sensitivity and specificity with serum samples; however, sensitivity in oral fluid samples was only 81%. By modifying the HCV 3.0 ELISA to utilize a secondary antibody cocktail that recognizes not only IgG but IgA and IgM as well, we attained 100% specificity and sensitivity with oral fluid samples.

'Catastrophic' antiphospholipid syndrome.

When 'catastrophic' is applied as an adjective to the antiphospholipid syndrome, it implies a characteristic presentation due to predominantly small blood vessel thrombosis leading to rapidly progressive failure of multiple organs and a frequently fatal outcome. We present the case of a 48-year-old woman who presented with the 'catastrophic' antiphospholipid syndrome without previous history of coagulation disorder or connective tissue disease that illustrates the difficulties in diagnosing and managing this disorder. We also review the factors that have been reported to have a role in the development of this condition and show how this case throws light on its pathogenesis.