Size-Controlled Synthesis of Gold Nanoparticles Tethering Antimicrobial Peptides with Potent Broad-Spectrum Antimicrobial and Antibiofilm Activities.

New antimicrobials are urgently needed to combat the rising global health concern of antibiotic resistance. Antimicrobial peptides (AMPs) are one of the leading candidates as new antimicrobials since they target bacterial membranes and are therefore less prone to bacterial resistance. However, poor enzymatic stability, high production costs, and toxicity are drawbacks that limit their clinical use. Conjugation of AMPs to gold nanoparticles (NPs) may help to improve enzymatic stability and, thus, their overall antimicrobial efficiency. We did a one-pot synthesis of size-controlled (10 nm) gold NPs selectively conjugated to lipopeptides and determined their antibacterial activity. The conjugates exhibited potent (0.13-1.25 µM) antimicrobial activity against clinical isolates, including Gram-positive methicillin-resistant Staphylococcus aureus (S. aureus) ATCC33593, Gram-negative Escherichia coli (E. coli) CTX-M-14, multidrug-resistant Pseudomonas aeruginosa LESB58 and Acinetobacter baumannii ATCC19606, and showed promising activity (90% inhibition of initial biofilms and 80% reduction of preformed biofilms) against S. aureus and E. coli DH5α biofilms at low micromolar concentrations. The conjugates were stable in rat serum and not toxic to representative mammalian cell lines in vitro (≤64 µM) and in vivo (≤100 µM).

Rapid detection of viable microbes with 5-cyano-2,3-di-(p-tolyl)tetrazolium chloride and 5(6)-carboxyfluorescein diacetate using a fibre fluorescence spectroscopy system.

AIMS: To assess the efficacy of two commercially available viability dyes, 5-cyano-2,3-di-(p-tolyl)tetrazolium chloride (CTC) and 5(6)-carboxyfluorescein diacetate (CFDA), in reporting on viable cell concentration and species using an all-fibre fluorometer. METHODS AND RESULTS: Four bacterial species (two Gram-positive and two Gram-negative) commonly associated with food poisoning or food spoilage (Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Bacillus cereus) were stained with CTC or CFDA and the fibre fluorometer was used to collect full fluorescence emission spectra. A good correlation between concentration and fluorescence intensity was found for Gram-negative bacteria between 107 and 108 colony-forming units (CFU) ml-1. There was no correlation with concentration for Gram-positive bacteria; however, the information in the CTC and CFDA spectra shows the potential to distinguish Gram-negative cells from Gram-positive cells, although it may simply reflect the overall bacterial metabolic activity under staining conditions from this study. CONCLUSIONS: The limit of detection (LoD) is too high in the dip-probe approach for analysis; however, the development of an approach measuring the fluorescence of single cells may improve this limitation. The development of new bacteria-specific fluorogenic dyes may also address this limitation. The ability to differentiate bacteria using these dyes may add value to measurements made to enumerate bacteria using CTC and CFDA.

Homo and Hetero-Branched Lipopeptide Dendrimers: Synthesis and Antimicrobial Activity.

Di-branched and tetra-branched versions of a previously reported analogue of the lipopeptide battacin were successfully synthesised using thiol-maleimide click and 1, 2, 3-triazole click chemistry. Antimicrobial studies against drug resistant clinical isolates of Escherichia coli (ESBL E. coli Ctx-M14), Pseudomonas aeruginosa (P. aeruginosa Q502), and Methicillin resistant Staphylococcus aureus (MRSA ATCC 33593), as well as clinically isolated Acinetobacter baumannii (A. baumannii ATCC 19606), and P. aeruginosa (ATCC 27853), revealed that the dendrimeric peptides have antimicrobial activity in the low micromolar range (0.5 -- 4 µM) which was 10 times more potent than the monomer peptides. Under high salt concentrations (150 mM NaCl, 2 mM MgCl2, and 2.5 mM CaCl2) the di-branched lipopeptides retained their antimicrobial activity while the monomer peptides were not active (>100 µM). The di-branched triazole click lipopeptide, Peptide 12, was membrane lytic, showed faster killing kinetics, and exhibited antibiofilm activity against A. baumannii and MRSA and eradicated > 85 % preformed biofilms at low micromolar concentrations. The di-branched analogues were > 30-fold potent than the monomers against Candida albicans. Peptide 12 was not haemolytic (HC10 = 932.12 µM) and showed up to 40-fold higher selectivity against bacteria and fungi than the monomer peptide. Peptide 12 exhibited strong proteolytic stability (>80 % not degraded) in rat serum over 24 h whereas > 95 % of the thiol-maleimide analogue (Peptide 10) was degraded. The tetra-branched peptides showed comparable antibacterial potency to the di-branched analogues. These findings indicate that dual branching using triazole click chemistry is a promising strategy to improve the antimicrobial activity and proteolytic stability of battacin based lipopeptides. The information gathered can be used to build effective antimicrobial dendrimeric peptides as new peptide antibiotics.

Prophylactic use of a lidocaine constant rate infusion versus saline in dogs undergoing balloon valvuloplasty for management of pulmonic stenosis: A randomized control trial.

OBJECTIVE: To evaluate the effect of a prophylactic lidocaine constant rate infusion (CRI) on the incidence and malignancy of catheter-induced ventricular ectopic complexes (VECs) during balloon valvuloplasty for management of pulmonic stenosis in dogs. STUDY DESIGN: Single-centre, prospective, randomized study. ANIMALS: Client-owned dogs (n = 70) with pulmonic stenosis. METHODS: Dogs were randomly assigned to one of two anaesthetic protocols: administration of lidocaine 2 mg kg-1 bolus followed by a CRI (50 µg kg-1 minute-1; group LD) or a saline placebo (group SL) during balloon valvuloplasty. All dogs were premedicated with methadone (0.3 mg kg-1) intramuscularly and a digital three-lead Holter monitor was applied. Anaesthetic co-induction was performed with administration of alfaxalone (2 mg kg-1) and diazepam (0.4 mg kg-1), and anaesthesia was maintained with isoflurane vaporised in 100% oxygen. CRIs were started on positioning of the dog in theatre and discontinued as the last vascular catheter was removed from the heart. All dogs recovered well and were discharged 24 hours postoperatively. Blinded Holter analysis was performed by an external veterinary cardiologist using commercially available dedicated analysis software; p < 0.05. RESULTS: Of the 70 dogs enrolled in the study, 61 were included in the final analysis: 31 in group LD and 30 in group SL. There was no significant difference between sinus beats (p = 0.227) or VECs (p = 0.519) between groups. In group LD, 19/31 (61.3%) dogs had a maximum ventricular rate ≥250 units and 20/30 (66.7%) dogs in group SL (p = 0.791). CONCLUSION AND CLINICAL RELEVANCE: In this study, the use of a prophylactic lidocaine bolus followed by CRI in dogs undergoing balloon valvuloplasty for management of pulmonic stenosis did not significantly decrease the incidence nor the malignancy of VECs during right heart catheterization compared with a saline CRI.

DETERMINATION OF ECHOCARDIOGRAPHIC TECHNIQUES FOR CLINICAL ASSESSMENT OF GALAPAGOS (CHELONOIDIS NIGRA COMPLEX) AND ALDABRA (ALDABRACHELYS GIGANTEA) TORTOISES.

The first objective of this study was to establish clinically relevant techniques for cardiac echocardiography in nonanesthetized Galapagos (Chelonoidis nigra complex) and Aldabra (Aldabrachelys gigantea) tortoises. A second objective was to establish guidelines for determining normal echocardiographic anatomy and function in both species. Select echocardiographic reference values were defined for 17 healthy Galapagos tortoises and 27 healthy Aldabra tortoises. Tortoises were either placed in ventral recumbency on an elevated surface or allowed to stand in a natural position by using food distraction. An ultrasound probe was applied in the left or right cervicobrachial window and was positioned in two long axis views to evaluate the three chambers of the heart and the associated great vessels, the presence of pericardial effusion, the atrioventricular inflow velocities, and pulmonic and aortic outflow velocities. The heart rate was 28 ± 12 (median ± SD) bpm, and the ejection fraction was 60.5 ± 10%. Thirty-four of 44 tortoises had identifiable physiologic pericardial effusion. All tortoises were successfully imaged using the techniques described, with consistent identification of cardiac structure and assessment of function. This study provides echocardiographic reference intervals for the clinical evaluation of suspected cardiac disease in captive-managed Galapagos and Aldabra tortoises.

Gut microbiota modulates COPD pathogenesis: role of anti-inflammatory Parabacteroides goldsteinii lipopolysaccharide.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a global disease characterised by chronic obstruction of lung airflow interfering with normal breathing. Although the microbiota of respiratory tract is established to be associated with COPD, the causality of gut microbiota in COPD development is not yet established. We aimed to address the connection between gut microbiota composition and lung COPD development, and characterise bacteria and their derived active components for COPD amelioration. DESIGN: A murine cigarette smoking (CS)-based model of COPD and strategies evaluating causal effects of microbiota were performed. Gut microbiota structure was analysed, followed by isolation of target bacterium. Single cell RNA sequencing, together with sera metabolomics analyses were performed to identify host responsive molecules. Bacteria derived active component was isolated, followed by functional assays. RESULTS: Gut microbiota composition significantly affects CS-induced COPD development, and faecal microbiota transplantation restores COPD pathogenesis. A commensal bacterium Parabacteroides goldsteinii was isolated and shown to ameliorate COPD. Reduction of intestinal inflammation and enhancement of cellular mitochondrial and ribosomal activities in colon, systematic restoration of aberrant host amino acids metabolism in sera, and inhibition of lung inflammations act as the important COPD ameliorative mechanisms. Besides, the lipopolysaccharide derived from P. goldsteinii is anti-inflammatory, and significantly ameliorates COPD by acting as an antagonist of toll-like receptor 4 signalling pathway. CONCLUSION: The gut microbiota-lung COPD axis was connected. A potentially benefial bacterial strain and its functional component may be developed and used as alternative agents for COPD prevention or treatment.

The antibacterial potency and antibacterial mechanism of a commercially available surface-anchoring quaternary ammonium salt (SAQAS)-based biocide in vitro.

AIMS: To determine the antimicrobial potency of a surface-anchored quaternary ammonium salt (SAQAS)-based biocide during in vitro wet and dry fomite assays and to determine the mechanism of killing bacteria on the surface. METHODS AND RESULTS: Wet and dry fomite assays were established in vitro for a commercially available biocide (SAQAS-A) applied to glass and low-density polyethylene (LDPE) surfaces. Both wet and dry fomite tests showed the active killing of Gram-positive and Gram-negative bacteria but not endospores. Assays measuring membrane permeability (ATP and DNA release), bacterial membrane potential and bacterial ROS production were correlated with the time-to-kill profiles to show SAQAS-A activity in suspension and applied to a surface. CONCLUSIONS: SAQAS-A is an effective biocide against model strains of vegetative bacteria. The killing mechanism for SAQAS-A observed minimal membrane depolarization, a surge in ROS production and assessment of membrane permeability supported the puncture of cells in both suspension and surface attachment, leading to cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: SAQAS represents effective surface biocides against single challenges with bacteria through a mechanical killing ability that supports real-world application if their durability can be demonstrated to maintain residual activity.

Association between delays to patient admission from the emergency department and all-cause 30-day mortality.

BACKGROUND: Delays to timely admission from emergency departments (EDs) are known to harm patients. OBJECTIVE: To assess and quantify the increased risk of death resulting from delays to inpatient admission from EDs, using Hospital Episode Statistics and Office of National Statistics data in England. METHODS: A cross-sectional, retrospective observational study was carried out of patients admitted from every type 1 (major) ED in England between April 2016 and March 2018. The primary outcome was death from all causes within 30 days of admission. Observed mortality was compared with expected mortality, as calculated using a logistic regression model to adjust for sex, age, deprivation, comorbidities, hour of day, month, previous ED attendances/emergency admissions and crowding in the department at the time of the attendance. RESULTS: Between April 2016 and March 2018, 26 738 514 people attended an ED, with 7 472 480 patients admitted relating to 5 249 891 individual patients, who constituted the study's dataset. A total of 433 962 deaths occurred within 30 days. The overall crude 30-day mortality rate was 8.71% (95% CI 8.69% to 8.74%). A statistically significant linear increase in mortality was found from 5 hours after time of arrival at the ED up to 12 hours (when accurate data collection ceased) (p<0.001). The greatest change in the 30-day standardised mortality ratio was an 8% increase, occurring in the patient cohort that waited in the ED for more than 6 to 8 hours from the time of arrival. CONCLUSIONS: Delays to hospital inpatient admission for patients in excess of 5 hours from time of arrival at the ED are associated with an increase in all-cause 30-day mortality. Between 5 and 12 hours, delays cause a predictable dose-response effect. For every 82 admitted patients whose time to inpatient bed transfer is delayed beyond 6 to 8 hours from time of arrival at the ED, there is one extra death.

The complex, bidirectional role of extracellular vesicles in infection.

Cells from all domains of life release extracellular vesicles (EVs), packages that carry a cargo of molecules that participate in communication, co-ordination of population behaviours, virulence and immune response mechanisms. Mammalian EVs play an increasingly recognised role to fight infection, yet may also be commandeered to disseminate pathogens and enhance infection. EVs released by bacterial pathogens may deliver toxins to host cells, signalling molecules and new DNA to other bacteria, and act as decoys, protecting infecting bacteria from immune killing. In this review, we explore the role of EVs in infection from the perspective of both the pathogen and host, and highlight their importance in the host/pathogen relationship. We highlight proposed strategies for EVs in therapeutics, and call attention to areas where existing knowledge and evidence is lacking.

A novel optical biosensor for in situ and small-scale monitoring of bacterial transport in saturated columns.

In situ monitoring techniques can provide new insight into bacterial transport after inoculating exogenous bacteria into contaminated soils for bioremediation. A real-time and non-destructive optical sensor (the optrode) was employed to monitor in situ transport of two fluorescently labelled bacteria - Green Fluorescent Protein (Gfp)-labelled, hydrophilic Pseudomonas putida and Tomato Fluorescent Protein (td)-labelled, hydrophobic Rhodococcus erythropolis, in a saturated sand column with and without rhamnolipid surfactant. In situ measurements were made at three sampling ports in the column with the optrode in two sets of column experiments. In Experiment 1, liquid samples were extracted for ex situ analyses (plate counts and fluorescence), while in Experiment 2 no liquid samples were extracted. Extracting liquid samples for ex situ analyses in Experiment 1 disturbed in situ measurements; in situ measured bacterial concentrations were lower, or a significant lag in breakthrough occurred relative to ex situ measurements. In Experiment 2, the optrode worked well in monitoring bacterial transport, which gave consistent transport parameters at each sampling port. Moreover, the optrode enabled the impact of bacterial hydrophobicity and rhamnolipid surfactant on bacterial transport to be observed. Specifically, hydrophilic P. putida was transported faster through the column than hydrophobic R. erythropolis; we infer from this result that fewer P. putida cells adsorb to sand particles than do R. erythropolis cells. The rhamnolipid surfactant enhanced the transport of both hydrophilic and hydrophobic bacteria. These two observations are consistent with Lifshitz-van der Waals forces and acid-base interactions between bacteria and sand.

Epipyrone A, a Broad-Spectrum Antifungal Compound Produced by Epicoccum nigrum ICMP 19927.

We have isolated a filamentous fungus that actively secretes a pigmented exudate when growing on agar plates. The fungus was identified as being a strain of Epicoccum nigrum. The fungal exudate presented strong antifungal activity against both yeasts and filamentous fungi, and inhibited the germination of fungal spores. The chemical characterization of the exudate showed that the pigmented molecule presenting antifungal activity is the disalt of epipyrone A-a water-soluble polyene metabolite with a molecular mass of 612.29 and maximal UV-Vis absorbance at 428 nm. This antifungal compound showed excellent stability to different temperatures and neutral to alkaline pH.

ECHOCARDIOGRAPHIC VARIABLES IN THE AFRICAN PAINTED DOG (LYCAON PICTUS).

The aim of this study was to screen for cardiac disease and report normal echocardiographic variables from a managed population of the African painted dog (Lycaon pictus; APD). The study included six male adult APDs under managed care with no evidence of significant structural cardiac disease. Echocardiography was performed with APD maintained under anesthesia with a combination of midazolam, butorphanol, and inhaled isoflurane. All animals underwent a full physical examination and two-dimensional (2D), M-mode, and Doppler echocardiography using standard views. Left ventricular volumes were calculated using the Simpson's method of discs. Medians with ranges were reported for the echocardiographic variables. No valvular structural abnormalities were noted. Trace mitral regurgitation was documented in five of six APD, trace tricuspid regurgitation in three of six, and mild pulmonic regurgitation in all APD. No aortic regurgitation was noted. Standard M-mode measurements fell within the normal ranges for allometrically scaled M-mode cardiac measurements for normal adult domestic dogs (Canis familiaris). Two-dimensional measurements were comparable with that of domestic dogs with similar body weight. The study demonstrates a similarity in cardiac size and function compared with normal adult domestic dogs and provides baseline echocardiographic variables for investigation of cardiac health in other populations of APDs in the future.

Induction of Microbial Oxidative Stress as a New Strategy to Enhance the Enzymatic Degradation of Organic Micropollutants in Synthetic Wastewater.

Organic micropollutants (OMPs) are pervasive anthropogenic contaminants of receiving waters where they can induce various adverse effects to aquatic life. Their ubiquitous environmental occurrence is primarily attributed to discharge from wastewater treatment plants due to incomplete removal by common biological wastewater treatment processes. Here, we assess a new strategy for promoting the degradation of six representative OMPs (i.e., sulfamethoxazole, carbamazepine, tylosin, atrazine, naproxen, and ibuprofen) by intentionally stimulating the production of microbial oxidoreductases to counter oxidative stress caused by oxygen perturbations. Mixed microbial cultures from a dairy farm wastewater were subjected to cyclic perturbations of dissolved oxygen (DO). A distance-based redundancy analysis was used to show that DO perturbations correlate with the abundance of Pseudomonadaceae and Rhodocyclaceae families, activities of peroxidases and cytochromes, and the degradation of OMPs. DO perturbation of 0.25 and 0.5 cycles/h led to most abundance of Pseudomonadaceae and Rhodocyclaceae families, showed higher activity of peroxidase and cytochrome, and gave largest removal of OMPs (removal of 92 ± 3% for sulfamethoxazole, 84 ± 3% for naproxen, 82 ± 3% for ibuprofen, 66 ± 2% for carbamazepine, 57 ± 15% for tylosin, and 88 ± 1% for atrazine).

Rapid and cost-effective evaluation of bacterial viability using fluorescence spectroscopy.

A rapid and easy method that takes advantage of an inexpensive and portable fibre-based spectroscopic system (optrode) to determine the ratio of live to dead bacteria is proposed. Mixtures of live and dead Escherichia coli with proportions of live:dead cells varying from 0 to 100% were stained using SYTO 9 and propidium iodide (PI) and measured using the optrode. We demonstrated several approaches to obtaining the proportions of live:dead E. coli in a mixture of both live and dead, from analyses of the fluorescence spectra collected by the optrode. To find a suitable technique for predicting the percentage of live bacteria in a sample, four analysis methods were assessed and compared: SYTO 9:PI fluorescence intensity ratio, an adjusted fluorescence intensity ratio, single-spectrum support vector regression (SVR) and multi-spectra SVR. Of the four analysis methods, multi-spectra SVR obtained the most reliable results and was able to predict the percentage of live bacteria in 108 bacteria/mL samples between c. 7 and 100% live, and in 107 bacteria/mL samples between c. 7 and 73% live. By demonstrating the use of multi-spectra SVR and the optrode to monitor E. coli viability, we raise points of consideration for spectroscopic analysis of SYTO 9 and PI and aim to lay the foundation for future work that uses similar methods for different bacterial species.

Molecular Weight and Charge Density Effects of Guanidinylated Biodegradable Polycarbonates on Antimicrobial Activity and Selectivity.

Six guanidine functionalized aliphatic biodegradable polycarbonates with varying molecular weights and charge densities were synthesized via postsynthesis modification of alkyne containing polycarbonates using Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry. The concept of passive diluting group was to modify the cationic charge density of the polycarbonate without changing its hydrophilicity. Within the molecular weight range from 8000 to 30000 g mol-1, these guanidine polycarbonates exhibited broad-spectrum biocidal activity with low toxicity to red blood cells (RBCs). The lowest molecular weight homopolymer sample (PG-8k-100) showed the best antimicrobial activity (MIC = 40 µg/mL against Escherichia coli and MIC = 20 µg/mL against Staphylococcus epidermidis) and least RBC toxicity (0.6% hemolysis at MIC). Within the three guanidine charge densities from 20% to 70%, the low to medium dilution samples (PG-8k-7030 and PG-8k-5050) had no obvious loss in antimicrobial activities compared to the nondiluted control sample PG-8k-100. However, upon further dilution, PG-8k-2080 gave the lowest antimicrobial activity.

Variation in laparoscopic anti-reflux surgery across England: a 5-year review.

BACKGROUND: Laparoscopic anti-reflux surgery (LARS) remains central to the management of gastro-oesophageal reflux disease but the scale and variation in provision in England is unknown. The aims of this study were firstly to examine the processes and outcomes of anti-reflux surgery in England and compare them to national guidelines and secondly to explore potential variations in practice nationally and establish peer benchmarks. METHODS: All adult patients who underwent LARSin England during the Financial years FY 2011/2012-FY 2016/2017 were identified in the Surgeon's Workload Outcomes and Research Database (SWORD), which is based on the Hospital Episode Statistics (HES) data warehouse. Outcomes included activity volume, day-case rate, short-stay rate, 2- and 30-day readmission rates and 30-day re-operation rates. Funnel plots were used to identify national variation in practice. RESULTS: In total, 12,086 patients underwent LARS in England during the study period. The operation rate decreased slightly over the study period from 5.2 to 4.6 per 100,000 people. Most outcomes were in line with national guidelines including the conversion rate (0.76%), 30-day re-operation rate (1.43%) and 2- and 30-day readmission rates (1.65 and 8.54%, respectively). The day-case rate was low but increased from 7.4 to 15.1% during the 5-year period. Significant variation was found, particularly in terms of hospital volume, and day-case, short-stay and conversion rates. CONCLUSION: Although overall outcomes are comparable to studies from other countries, there is significant variation in anti-reflux surgery activity and outcomes in England. We recommend that units use these data to drive local quality improvement efforts.

Profiling of headspace volatiles from Escherichia coli cultures using silicone-based sorptive media and thermal desorption GC-MS.

Headspace sorptive extraction technique using silicone based sorptive media coated stir bars is used for the first time here to extract, identify, and quantify heavy volatile organic compounds present in Escherichia coli culture headspace. Detection of infection presence is largely accomplished in laboratories through physical sampling and subsequent growth of cultures for biochemical testing. The use of volatile biomarkers released from pathogens as indicators for pathogenic presence can vastly reduce the time needed whilst improving the success rates for infection detection. To validate this, by using a contactless headspace sorptive extraction technique, the volatile compounds released from E. coli, grown in vitro, have been extracted and identified. Two different sorptive media for extracting these headspace volatiles were compared in this study and the identified volatiles were quantified. The large phase volume and wider retention of this sorptive technique compared to traditional sampling approach enabled preconcentration and collection of wider range of volatiles towards developing an extensive database of such heavy volatiles associated with E. coli. This supplements the existing data of potential bacterial markers and use of internal standards in these tests allows semi-quantitative estimation of these compounds towards the development and optimization of novel pathogen sensing devices.

Identification of an immune modulation locus utilising a bovine mammary gland infection challenge model.

Inflammation of the mammary gland following bacterial infection, commonly known as mastitis, affects all mammalian species. Although the aetiology and epidemiology of mastitis in the dairy cow are well described, the genetic factors mediating resistance to mammary gland infection are not well known, due in part to the difficulty in obtaining robust phenotypic information from sufficiently large numbers of individuals. To address this problem, an experimental mammary gland infection experiment was undertaken, using a Friesian-Jersey cross breed F2 herd. A total of 604 animals received an intramammary infusion of Streptococcus uberis in one gland, and the clinical response over 13 milkings was used for linkage mapping and genome-wide association analysis. A quantitative trait locus (QTL) was detected on bovine chromosome 11 for clinical mastitis status using micro-satellite and Affymetrix 10 K SNP markers, and then exome and genome sequence data used from the six F1 sires of the experimental animals to examine this region in more detail. A total of 485 sequence variants were typed in the QTL interval, and association mapping using these and an additional 37 986 genome-wide markers from the Illumina SNP50 bovine SNP panel revealed association with markers encompassing the interleukin-1 gene cluster locus. This study highlights a region on bovine chromosome 11, consistent with earlier studies, as conferring resistance to experimentally induced mammary gland infection, and newly prioritises the IL1 gene cluster for further analysis in genetic resistance to mastitis.

Changes in estrogenicity and micropollutant concentrations across unit processes in a biological wastewater treatment system.

The behavior of 10 micropollutants, i.e. four estrogens (estrone, 17ß-estradiol, estriol, 17α-ethynylestradiol), carbamazepine (CBZ), sulfamethoxazole (SMX), triclosan, oxybenzone, 4-nonylphenol, and bisphenol A, was investigated in a typical domestic wastewater treatment plant. LC-MS and yeast estrogen screen bioassay were used to study the changes in micropollutants and estrogenicity across unit processes in the treatment system. Primary treatment via sedimentation showed that only 4-nonylphenol was removed, but led to no significant change in estrogenicity. Secondary treatment by the biological nitrification-dentrification process showed complete removal of oxybenzone and partial removal of the estrogens, which led to a decrease in estrogenic activity from 80 to 48 ng/L as estradiol equivalent (EEq). Ultraviolet treatment completely degraded the estrogens and triclosan, but failed to lower the concentrations of bisphenol A, SMX, and CBZ; a decrease in estrogenic activity from 48 to 5 ng/L EEq across the unit, a value that was only slightly larger than the observed EEq of 1 ng/L for the deionized control. Similarly, the anaerobic digestion of sludge completely degraded estrogens, oxybenzone, and SMX, but had no impact on bisphenol A, triclosan, and CBZ. The study emphasises the need to complement chemical analyses with estrogenic bioassays to evaluate the efficacy of waste water treatment plants.

A rapid and low-cost estimation of bacteria counts in solution using fluorescence spectroscopy.

The fluorescence spectrum of bacterially bound acridine orange (AO) was investigated to evaluate its use for the rapid enumeration of bacteria. Escherichia coli ATCC 25922 samples were stained with 2 × 10-2, 2 × 10-3 or 2 × 10-4% w/v AO, followed by 3, 2 or 0 washing cycles, respectively, and fluorescence spectra were recorded using a fibre-based spectroscopic system. Independent component analysis was used to analyse the spectral datasets for each staining method. Bacterial concentration order of magnitude classification models were calculated using independent component weights. The relationship between fluorescence intensity of bound AO and bacterial concentration was not linear. However, the spectral signals collected for AO stain concentration-bacterial concentration pairs were reproducible and unique enough to enable classification of samples. When above 105 CFU ml-1, it was possible to rapidly determine what the order of magnitude of bacterial concentration of a sample was using a combination of two of the sample preparation methods. A relatively inexpensive (around US$10 per test) rapid method (within 25 min of sampling) for enumeration of bacteria by order of magnitude will reduce the time and cost of microbiological tests requiring gross concentration information. Graphical Abstract Fluorescence spectra of bacterially bound acridine orange (AO) were used for the rapid enumeration of bacteria. Order of magnitude bacterial concentration classification models were calculated using independent components analysis of these fluorescence spectra. When above 105 CFU ml-1, it was possible to rapidly determine the order of magnitude of bacterial concentration of a sample using a combination of two sample preparation methods.