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Gram-negative bacteria possess numerous defenses against antibiotics, due to the intrinsic permeability barrier of their outer membrane (OM), explaining the recalcitrance of some common and life-threatening infections. We report the formulation of a new drug, PPA148, which shows promising activity against all Gram-negative bacteria included in the ESKAPEE pathogens. PPA148 was solubilized by inclusion complexation with cyclodextrin followed by encapsulation in liposomes. The complex and liposomal formulation presented increased activity against E. colicompared to the pure drug when assessed with the Kirby Bauer assay. The novel formulation containing 1 μg PPA148 reached similar efficacy levels equivalent to those of 30 μg of pure rifampicin. A range of biophysical techniques was used to explore the mechanism of drug uptake. Langmuir trough (LT) and neutron reflectivity (NR) techniques were employed to monitor the interactions between the drug and the formulation with model membranes. We found evidence for liposome fusion with the model Gram-negative outer membrane and for cyclodextrins acting as inner membrane (IM) permeation enhancers without presenting intrinsic antimicrobial activity. An antibiotic-in-cyclodextrin-in-liposomes (ACL) formulation was developed, which targets both the bacterial OM and IM, and offers promise as a means to breach the Gram-negative cell envelope.

Pulmonary symptoms in cystic fibrosis (CF) begin in early life with chronic lung infections and concomitant airway inflammation leading to progressive loss of lung function. Gradual pulmonary function decline is interspersed with periods of acute worsening of respiratory symptoms known as CF pulmonary exacerbations (CFPEs). Cumulatively, CFPEs are associated with more rapid disease progression. In this study multiple sputum samples were collected from adult CF patients over the course of CFPEs to better understand how changes in microbiota are associated with CFPE onset and management. Data were divided into five clinical periods: pre-CFPE baseline, CFPE, antibiotic treatment, recovery, and post-CFPE baseline. Samples were treated with propidium monoazide prior to DNA extraction, to remove the impact of bacterial cell death artefacts following antibiotic treatment, and then characterised by 16S rRNA gene-targeted high-throughput sequencing. Partitioning CF microbiota into core and rare groups revealed compositional resistance to CFPE and resilience to antibiotics interventions. Mixed effects modelling of core microbiota members revealed no significant negative impact on the relative abundance of Pseudomonas aeruginosa across the exacerbation cycle. Our findings have implications for current CFPE management strategies, supporting reassessment of existing antimicrobial treatment regimens, as antimicrobial resistance by pathogens and other members of the microbiota may be significant contributing factors.

Pulmonary symptoms in cystic fibrosis (CF) begin in early life with chronic lung infections and concomitant airway inflammation leading to progressive loss of lung function. Gradual pulmonary function decline is interspersed with periods of acute worsening of respiratory symptoms known as CF pulmonary exacerbations (CFPEs). Cumulatively, CFPEs are associated with more rapid disease progression. In this study multiple sputum samples were collected from adult CF patients over the course of CFPEs to better understand how changes in microbiota are associated with CFPE onset and management. Data were divided into five clinical periods: pre-CFPE baseline, CFPE, antibiotic treatment, recovery, and post-CFPE baseline. Samples were treated with propidium monoazide prior to DNA extraction, to remove the impact of bacterial cell death artefacts following antibiotic treatment, and then characterised by 16S rRNA gene-targeted high-throughput sequencing. Partitioning CF microbiota into core and rare groups revealed compositional resistance to CFPE and resilience to antibiotics interventions. Mixed effects modelling of core microbiota members revealed no significant negative impact on the relative abundance of Pseudomonas aeruginosaacross the exacerbation cycle. Our findings have implications for current CFPE management strategies, supporting reassessment of existing antimicrobial treatment regimens, as antimicrobial resistance by pathogens and other members of the microbiota may be significant contributing factors.

Long-term macrolide treatment has proven benefit in inflammatory airways diseases, but whether it leads to changes in the composition of respiratory microbiota is unknown. We aimed to assess whether long-term, low-dose erythromycin treatment changes the composition of respiratory microbiota in people with non-cystic fibrosis bronchiectasis.

ABSTRACTSpontaneously expectorated sputum is traditionally used as the sampling method for the investigation of lower airway infections. While guidelines exist for the handling of these samples for culture-based diagnostic microbiology, there is no comparable consensus on their handling prior to culture-independent analysis. The increasing incorporation of culture-independent approaches in diagnostic microbiology means that it is of critical importance to assess potential biases. The aim of this study was to assess the impact of delayed freezing on culture-independent microbiological analyses and to identify acceptable parameters for sample handling. Sputum samples from eight adult cystic fibrosis (CF) patients were collected and aliquoted into sterile Bijou bottles. Aliquots were stored at room temperature before being frozen at -80°C for increasing intervals, up to a 72-h period. Samples were treated with propidium monoazide to distinguish live from dead cells prior to DNA extraction, and 16S rRNA gene pyrosequencing was used to characterize their bacterial compositions. Substantial variation was observed in samples with high-diversity bacterial communities over time, whereas little variation was observed in low-diversity communities dominated by recognized CF pathogens, regardless of time to freezing. Partitioning into common and rare species demonstrated that the rare species drove changes in similarity. The percentage abundance of anaerobes over the study significantly decreased after 12 h at room temperature (P= 0.008). Failure to stabilize samples at -80°C within 12 h of collection results in significant changes in the detected community composition.

ABSTRACTPrimary ciliary dyskinesia (PCD) is a genetic disease characterized by abnormalities in ciliary function, leading to compromised airway clearance and chronic bacterial infection of the upper and lower airways. The compositions of these infections and the relationships between their characteristics and disease presentation are poorly defined. We describe here the first systematic culture-independent evaluation of lower airway bacteriology in PCD. Thirty-three airway samples (26 from sputum, 7 from bronchoalveolar lavage [BAL] fluid) were collected from 24 PCD patients aged 4 to 73 years. 16S rRNA quantitative PCR and pyrosequencing were used to determine the bacterial loads and community compositions of the samples. Bacterial loads, which ranged from 1.3 × 104to 5.2 × 109CFU/ml, were positively correlated with age (P= 0.002) but not lung function. An analysis of ~7,000 16S rRNA sequences per sample identified bacterial species belonging to 128 genera. The concurrently collected paired samples showed high bacterial community similarity. The mean relative abundance of the dominant genera was 64.5% (standard deviation [SD], 24.5), including taxa reported through standard diagnostic microbiology (members of the genera Pseudomonas, Haemophilus, and Streptococcus) and those requiring specific ex vivogrowth conditions (members of the genera Prevotellaand Porphyromonas). The significant correlations observed included a positive relationship between Pseudomonas aeruginosarelative abundance and age and a negative relationship between P. aeruginosarelative abundance and lung function. Members of the genus Ralstoniawere also found to contribute substantially to the bacterial communities in a number of patients. Follow-up samples from a subset of patients revealed high levels of bacterial community temporal stability. The detailed microbiological characterization presented here provides a basis for the reassessment of the clinical management of PCD airway infections.

ABSTRACTThe aim of this study was to determine whether geographical differences impact the composition of bacterial communities present in the airways of cystic fibrosis (CF) patients attending CF centers in the United States or United Kingdom. Thirty-eight patients were matched on the basis of clinical parameters into 19 pairs comprised of one U.S. and one United Kingdom patient. Analysis was performed to determine what, if any, bacterial correlates could be identified. Two culture-independent strategies were used: terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA clone sequencing. Overall, 73 different terminal restriction fragment lengths were detected, ranging from 2 to 10 for U.S. and 2 to 15 for United Kingdom patients. The statistical analysis of T-RFLP data indicated that patient pairing was successful and revealed substantial transatlantic similarities in the bacterial communities. A small number of bands was present in the vast majority of patients in both locations, indicating that these are species common to the CF lung. Clone sequence analysis also revealed that a number of species not traditionally associated with the CF lung were present in both sample groups. The species number per sample was similar, but differences in species presence were observed between sample groups. Cluster analysis revealed geographical differences in bacterial presence and relative species abundance. Overall, the U.S. samples showed tighter clustering with each other compared to that of United Kingdom samples, which may reflect the lower diversity detected in the U.S. sample group. The impact of cross-infection and biogeography is considered, and the implications for treating CF lung infections also are discussed.

The role of infection in bronchopulmonary dysplasia (BPD) is unknown. We present an observational study of 55 premature infants born weighing less than 1.3 kg within two level III neonatal intensive care units. Endotracheal aspirates (ETA) and nasogastric aspirates (NGA) were studied with denaturing gradient gel electrophoresis (DGGE) profiling to elucidate the total bacterial community, and species-specific PCR was used to detect the presence of Mycoplasmahominis, Ureaplasma urealyticum, and Ureaplasma parvum. DGGE identified bacterial species in 59% of NGA and ETA samples combined. A diverse range of species were identified including several implicated in preterm labor. Species-specific PCR identified M.hominisin 25% of NGA and 11% of ETA samples. Among the 48 infants surviving up to 36 wk-postconceptual age, ordinal logistic regression showed the odds ratio for BPD or death where Ureaplasmawas present/absent as 4.80 (95% CI 1.15–20.13). After adjusting for number of days ventilated, this was reduced to 2.04 (0.41–10.25). These data demonstrate how the combined use of DGGE and species-specific PCR identifies a high exposure in uteroand around the time of birth to bacteria that might be causally related to preterm delivery and subsequent lung injury.

The role of infection in bronchopulmonary dysplasia (BPD) is unknown. We present an observational study of 55 premature infants born weighing less than 1.3 kg within two level III neonatal intensive care units. Endotracheal aspirates (ETA) and nasogastric aspirates (NGA) were studied with denaturing gradient gel electrophoresis (DGGE) profiling to elucidate the total bacterial community, and species-specific PCR was used to detect the presence of Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum. DGGE identified bacterial species in 59% of NGA and ETA samples combined. A diverse range of species were identified including several implicated in preterm labor. Species-specific PCR identified M. hominis in 25% of NGA and 11% of ETA samples. Among the 48 infants surviving up to 36 wk-postconceptual age, ordinal logistic regression showed the odds ratio for BPD or death where Ureaplasma was present/absent as 4.80 (95% CI 1.15–20.13). After adjusting for number of days ventilated, this was reduced to 2.04 (0.41–10.25). These data demonstrate how the combined use of DGGE and species-specific PCR identifies a high exposure in utero and around the time of birth to bacteria that might be causally related to preterm delivery and subsequent lung injury.

In recent articles (Rogers et al., 2009, Rogers et al., 2010), we discussed a fundamental shift in the way in which polymicrobial infections can be viewed. In these articles, we used chronic bacterial infections of the lower airways, and specifically those that occur in cystic fibrosis (CF) patients, as a model system. These infections are of course critical in clinical terms for these patients; respiratory failure due to a combination of these chronic infections with the host immune response that they elicit remains the leading cause of mortality in CF. Given the importance of maintaining lung health in these patients, the CF airways are the focus of a wide range of scientific and clinical studies. In particular, research momentum has built in relation to identifying the microbes present in the CF lung. Already, many important insights have been gained through the application of increasingly sophisticated culture-independent analytical methodologies to identify all microbial nucleic acids (Bittar et al., 2008, Ecker et al., 2008, Rogers et al., 2004, Sibley et al., 2008b) and those from viable or metabolically active microbes (Rogers et al., 2005, Rogers et al., 2008) in the CF lung. In doing so, the data generated have revealed microbial assemblages of far greater diversity and in turn complexity than has previously been recognised in this context. These studies, which have served to highlight the inadequacy of traditional culture-based diagnostic microbiology to fully characterise such infections (Rogers et al., 2009), have also led to a significant shift in our view of what the word "infection" represents for these and other chronic diseases, with potentially important implications for their optimal treatment. In this article we contrast the information we and others have accrued from chronic lung infections with data generated from studies examining the microbial communities present in the gut. In doing so we highlight parallels between these two contexts and discuss how these commonalities can inform clinical thinking.

ABSTRACTSampling of the lower airways of the adult cystic fibrosis (CF) lung has received insufficient detailed consideration, with the importance of sampling strategies for bacteriological outcome not known. Although spontaneously expectorated sputum (SES) samples are often used for diagnostic bacteriological analysis, induced sputum (IS) methods have advantages. This study examined whether significant differences in bacterial content were detected when using a culture-independent, molecular profiling technique to analyze SES or IS samples. Moreover, this work examined what trends relating to bacterial species distributions and reproducibility were found in sequentially induced sputum samples and what implications this has for pathogen detection. Terminal restriction fragment length polymorphism (T-RFLP) analysis was performed on a SES sample and 4 subsequent IS samples taken at 5-min intervals from 10 clinically stable, adult CF patients. This was repeated over 3 sampling days, with variability between samples, induction periods, and sampling days determined. A diverse range of bacterial species, including potentially novel pathogens, was found. No significant difference in bacterial content was observed for either SES or serial IS samples. On average, the analysis of a single sample from any time point resolved ~58% of total bacterial diversity achieved by analysis of an SES sample and 4 subsequent IS samples. The reliance on analysis of a single respiratory sample was not sufficient for the detection of recognized CF pathogens in all instances. Close correlation between T-RFLP and microbiological data in the detection of key species indicates the importance of these findings in routine diagnostics for the detection of recognized and novel CF pathogens.

The article reports on the historical significance of the ancient Angus building Restenneth Priory located in Forfar, Scotland. The Battle of Nechtansmere, which was fought between the Picts and the Angles took place in this area in 685 AD. The first church at Restenneth was erected in 710 AD on this ancient religious site. Bishop of Saint Andrews David de Bernham completed and consecrated the chancel of Restenneth Priory in August 30, 1243 AD.

Objectives: To review the main phenomenological variants observed among bulimia spectrum syndromes and the factors believed to act etiologically for them and also to generate an etiologic model that accommodates known heterogeneities within the population suffering bulimic syndromes.Method: Defining bulimic syndromes broadly, we address threshold and subthreshold forms of bulimia nervosa (BN) and also the provisional or newly proposed diagnoses of binge eating disorder (BED) and purging disorder (PD). We review evidence bearing on the validity of these diagnostic entities and on the place of sociocultural, family-developmental, neurobiological, and genetic factors in a multidimensional etiologic model for these classifications.Results: Available data validate certain bulimic phenotypes and subphenotypes that are characterized by such traits as impulsivity or affective instability. Findings associate subphenotypic, or trait-based, variations with putative endophenotypes, such as reduced serotonin transporter activity, and with candidate genotypes affecting the serotonin system; the data also indicate intriguing correspondences between gene–environment interactions and subphenotypic variations along such dimensions as novelty seeking.Conclusions: Bulimic syndromes sometimes reflect a primary disruption of controls over mood, impulses, and appetite in individuals showing marked psychopathology; at other times, they reflect a more circumscribed erosion of appetitive controls in relatively intact individuals, following prolonged dieting. We argue that dimensional perspectives involving careful attention to comorbid personality traits and symptoms are needed to accommodate existing heterogeneities within the population suffering from bulimia and to characterize the etiologic roles of familial-developmental, neurobiological, and genetic variables (and of interactions among these variables) in bulimic syndromes.

Separate lines of research suggest that the functional alterations in the serotonin (5‐HT) 2A receptor are associated with 5‐HT tone, behavioral impulsiveness, and bulimia nervosa (BN). We explored the effect of allelic variations within the 5‐HT2A receptor gene promoter polymorphism −1438G/A on trait impulsiveness and serotonin function in women with BN. Participants included women with BN having the A allele (i.e., AA homozygotes and AG heterozygotes, BNA+, N = 21); women with BN but without the A allele (i.e., GG homozygotes, BNGG, N = 12), and normal eater control women having the A allele (NEA+, N = 19) or without the A allele (NEGG; N = 9). The women were assessed for psychopathological tendencies and eating disorder symptoms, and provided blood samples for measurement of serial prolactin responses following oral administration of the post‐synaptic partial 5‐HT agonist meta‐chlorophenylpiperazine (m‐CPP). The BNGG group had higher scores than the other groups on self‐report measures of non‐planning and overall impulsiveness and had blunted prolactin response following m‐CPP. The bulimic groups did not differ from each other on current eating symptoms or on frequencies of other Axis I mental disorders. Findings indicate that women with BN who are GG homozygotes on the −1438G/A promoter polymorphism are characterized by increased impulsiveness and lower sensitivity to post‐synaptic serotonin activation. These findings implicate the GG genotype in the co‐aggregation of impulsive behaviors and alterations of post‐synaptic 5‐HT functioning in women with BN. © 2005 Wiley‐Liss, Inc.

The presence of Class II transposon genes related to Tn21and Tn501, and their structural arrangements have been determined in a collection of 124 mercury resistant Gram‐negative bacteria. Seventy‐five of the 124 isolates contained a tnpA(transposase) gene related to Tn21and Tn501and in all 64 isolates that contained both tnpAgenes and plasmids, the tnpAgene was plasmid borne. The relative orientation of the tnpgenes and the meroperon (encoding mercury resistance) was also studied and revealed the presence of two distinct structural groups. The merCgene was present in 44 isolates. Five isolates were found to carry integrase genes and these contained inserted gene cassettes varying in size from 1.1 kb to 4.5 kb. The structural arrangement of the tnpAand tnpR(resolvase) genes within the isolates was determined. Sixty‐nine of the 75 tnpAcontaining isolates had an arrangement of tnpAand tnpRgenes similar to that found in the Tn21subgroup of transposons. Four strains did not produce a PCR product using tnpRprimers. The remaining two isolates had undetermined arrangements of tnpAand tnpRgenes. No Tn3‐like arrangements of tnpAand tnpRgenes were present in these isolates, despite being detected in DNA extracted directly from the isolation sites. This suggests that Tn3‐like arrangements of tnpAand tnpRgenes are not commonly associated with mercury resistance genes in these environments. It was also apparent that the recombination events which have previously been observed in these strains have not significantly affected the diversity of the transposon structures within the isolates.

Several studies have documented the retrograde facilitation of memory by alcohol, but the mechanisms responsible for this curious effect are unknown. In an experiment designed to complement previous studies on incidental learning, social drinkers (men aged 18 to 30; n= 44) took part in an experiment examining the effects of alcohol on intentional learning of emotionally salient verbal stimuli. Learning occurred when participants were sober. Alcohol or placebo (1.0 vs. 0.1 ml/kg) was consumed after learning, and memory was tested, sober, 24 hr later. Compared with placebo, alcohol modestly enhanced recall of positive but not negative stimuli. Furthermore, results suggest that the reinforcing effects on memory for positive (relative to negative) stimuli occurred in association with acute psychomotor stimulant effects during the ascending limb of the blood alcohol curve. The present finding that alcohol appeared to enhance intentional learning in association with its incentive effects contrasts results from previous studies, demonstrating that alcohol appears to enhance incidental learning by memory mechanisms independent of its incentive effects. These findings support a theory of alcoholism that is based on motivational systems.

Mercury and its compounds are distributed widely across the earth. Many of the chemical forms of mercury are toxic to all living organisms. However, bacteria have evolved mechanisms of resistance to several of these different chemical forms, and play a major role in the global cycling of mercury in the natural environment. Five mechanisms of resistance to mercury compounds have been identified, of which resistance to inorganic mercury (HgR) is the best understood, both in terms of the mechanisms of resistance to mercury and of resistances to heavy metals in general. Resistance to inorganic mercury is encoded by the genes of the meroperon, and can be located on transposons, plasmids and the bacterial chromosome. Such systems have a worldwide geographical distribution, and furthermore, are found across a wide range of both Gram‐negative and Gram‐positive bacteria from both natural and clinical environments. The presence of mergenes in bacteria from sediment cores suggest that meris an ancient system. Analysis of DNA sequences from meroperons and genes has revealed genetic variation both in operon structure and between individual genes from different meroperons, whilst analysis of bacteria which are sensitive to inorganic mercury has identified a number of vestigial non‐functional operons. It is hypothesised that mer, due to its ubiquity with respect to geographical location, environment and species range, is an ancient system, and that ancient bacteria carried genes conferring resistance to mercury in response to increased levels of mercury in natural environments, perhaps resulting from volcanic activity. Models for the evolution of both a basic meroperon and for the Tn21‐related family of meroperons and transposons are suggested. The study of evolution in bacteria has recently become dominated by the generation of phylogenies based on 16S rRNA genes. However, it is important not to underestimate the roles of horizontal gene transfer and recombinational events in evolution. In this respect meris a suitable system for evaluating phylogenetic methods which incorporate the effects of horizontal gene transfer. In addition, the meroperon provides a model system in the study of environmental microbiology which is useful both as an example of a genotype which is responsive to environmental pressures and as a generic tool for the development of new methodology for the analysis of bacterial communities in natural environments.

Scandals show that ethics is an important topic in financial planning. Our research analyses 212 financial ombudsman decisions (2013–2018) to understand the nature of financial planning misconduct in complaint decisions. We develop a coding structure to ascertain what professional conduct involves and then use content analysis and cluster analysis to identify the aspects of professional conduct occurring in these misconduct decisions. Diligence, acting in the client’s best interest and having no reasonable basis for advice are interconnected elements in over half of these decisions. Secondary elements are misleading statements, conflicts of interest and disclosure. Analysis of decisions involving fiduciary duty showed that financial planners failed to ascertain a client’s circumstances and did not form advice based on their client’s information. As financial planning professionalises, future research, financial planning education, policy and practice should address these issues.JEL Classification: D14, G20, G50

We recently reported that, among bulimic women, previously abused carriers of the 5HTTLPR S allele showed special propensities towards novelty seeking (implying recklessness or impulsivity) and interpersonal insecurity. We subsequently re‐analyzed our data, to examine the bearing of the 5HTTLPR polymorphism and prior sexual or physical maltreatment upon validated, higher‐order personality‐traits. Ninety women with bulimic syndromes were genotyped for 5HTTLPR “short” (S) and “long” (LG and LA) alleles, and then assessed for eating symptoms, history of sexual or physical abuse, and the higher‐order personality traits Emotional Dysregulation, Dissocial Behavior, Inhibition, and Compulsivity. With a classification based on a biallelic model of 5HTTLPR (i.e., presence or absence of at least one S‐allele copy), multiple regression indicated a significant proportion of variance in Dissocial Behavior to be explained by an abuse × genotype interaction—greater psychopathology occurring in abused S‐allele carriers. A parallel analysis applying a triallelic model of 5HTTLPR (i.e., presence or absence of at least one copy of presumably low‐function S or LG alleles) produced a similar pattern, but no statistically significant effect. The finding that bulimic 5HTTLPR S‐allele carriers who are previously abused display elevations on Dissocial Behavior corroborates previous observations concerning phenomenological correlates of traumatic stress in 5HTTLPR S allele carriers. © 2007 Wiley‐Liss, Inc.

A letter to the editor is presented telling about concerns regarding the extermination of red squirrel species in Scotland while there are still many grey squirrels and adding about the disappearing species of brown hare as well.

ABSTRACTRecent studies have greatly extended our understanding of the microbiota present in and on the human body. Here, advanced sequencing strategies have provided unprecedented analytical power. The important implications that the emerging data have for human health emphasize the need to intensify research in this area (D. A. Relman, Nature 486:194-195, 2012). It is already clear from these studies that the microbiotas characterized in different body locations of healthy individuals are both complex and diverse (The Human Microbiome Project Consortium, Nature 486:215-221). These studies also provide a point of contrast for investigations that aim to characterize the microbiota present in disease conditions. In this regard, Madan et al. (mBio 3(4):e00251-12, 2012) monitored the development over time of microbiota in the oropharynges and feces of neonates with cystic fibrosis and explored the potential for interactions between these complex microbial systems.

The diversity of resolvase (tnpR) genes carried by a number of mercury resistant soil bacteria has been investigated by DNA sequencing. The resulting DNA sequence information was compared to previously published tnpR. DNA sequences and to previously published restriction fragment length polymorphism (RFLP) data, permitting the relationships between DNA sequencing and RFLP approaches to be studied by the use of phylogenetic trees. DNA maximum likelihood and DNA parsimony were used to construct a variety of phylogenetic trees. DNA sequencing confirmed the validity of RFLP analysis and highlighted the importance of restriction endonuclease choice upon the resulting RFLP patterns and dendrogram topology. The tnpR genes of two previously uncharacterised mercury resistant bacteria, T2–7 and T2–12 were also studied. DNA sequence data placed T2–7 in a previously described gene class, tnpR-D and T2–12 in a new gene class, tnpR-F. The significance of this data with respect to the recombination and evolution events occurring within bacterial populations are discussed.