The carbon costs of global wood harvests.

After agriculture, wood harvest is the human activity that has most reduced the storage of carbon in vegetation and soils1,2. Although felled wood releases carbon to the atmosphere in various steps, the fact that growing trees absorb carbon has led to different carbon-accounting approaches for wood use, producing widely varying estimates of carbon costs. Many approaches give the impression of low, zero or even negative greenhouse gas emissions from wood harvests because, in different ways, they offset carbon losses from new harvests with carbon sequestration from growth of broad forest areas3,4. Attributing this sequestration to new harvests is inappropriate because this other forest growth would occur regardless of new harvests and typically results from agricultural abandonment, recovery from previous harvests and climate change itself. Nevertheless some papers count gross emissions annually, which assigns no value to the capacity of newly harvested forests to regrow and approach the carbon stocks of unharvested forests. Here we present results of a new model that uses time discounting to estimate the present and future carbon costs of global wood harvests under different scenarios. We find that forest harvests between 2010 and 2050 will probably have annualized carbon costs of 3.5-4.2 Gt CO2e yr-1, which approach common estimates of annual emissions from land-use change due to agricultural expansion. Our study suggests an underappreciated option to address climate change by reducing these costs.

Activity of a nitric oxide-generating wound treatment system against wound pathogen biofilms.

Wound bioburden plays an important role in impaired healing and development of infection-related complications. The objective of this study was to determine the efficacy of an innovative two-layer nitric oxide-generating system (NOx) to prevent and treat biofilms formed by bacterial and fungal pathogens commonly associated with wound infection, and activity against Pseudomonas aeruginosa virulence factors. Single- and mixed-species biofilms were grown for 24 h on nitrocellulose filters placed on agar. Filters were covered with either NOx or placebo, before and after biofilm formation. Populations of bacteria and yeasts were determined using viable counts. Pyocyanin and elastase production from P. aeruginosa were determined in supernatants derived from suspended biofilms. Efficacy of NOx was demonstrated against Staphylococcus aureus, P. aeruginosa, Acinetobacter baumannii, Escherichia coli and Candida spp. Population reductions between 2- and 10-log fold were observed. Pyocyanin and elastase activities from P. aeruginosa were reduced 1.9- and 3.2-fold, respectively. This study demonstrated activity of NOx against formation and treatment of single- and mixed-species biofilms, including multidrug-resistant strains. NOx represents a new generation of antimicrobial agent with potent, broad-spectrum activity, and with no evidence of resistance development.

Correction: Modulation of behaviour and virulence of a high alginate expressing Pseudomonas aeruginosa strain from cystic fibrosis by oral commensal bacterium Streptococcus anginosus.

[This corrects the article DOI: 10.1371/journal.pone.0173741.].

Modulation of behaviour and virulence of a high alginate expressing Pseudomonas aeruginosa strain from cystic fibrosis by oral commensal bacterium Streptococcus anginosus.

Cystic fibrosis (CF) airways harbour complex and dynamic polymicrobial communities that include many oral bacteria. Despite increased knowledge of CF airway microbiomes the interaction between established CF pathogens and other resident microbes and resulting impact on disease progression is poorly understood. Previous studies have demonstrated that oral commensal streptococci of the Anginosus group (AGS) can establish chronic pulmonary infections and become numerically dominant in CF sputa indicating that they play an important role in CF microbiome dynamics. In this study a strain of Pseudomonas aeruginosa (DWW2) of the mucoid alginate overproducing phenotype associated with chronic CF airway infection and a strain of the oral commensal AGS species Streptococcus anginosus (3a) from CF sputum were investigated for their ability to co-exist and their responses to biofilm co-culture. Bacteria in biofilms were quantified, pyocyanin expression by DWW2 was measured and the effect of AGS strain 3a on reversion of DWW2 to a non-mucoidal phenotype investigated. The virulence of DWW2, 3a and colony variant phenotypes of DWW2 in mono- and co-culture were compared in a Galleria mellonella infection model. Co-culture biofilms were formed in normoxic, hypercapnic (10% CO2) and anoxic atmospheres with the streptococcus increasing in number in co-culture, indicating that these bacteria would be able to co-exist and thrive within the heterogeneous microenvironments of the CF airway. The streptococcus caused increased pyocyanin expression by DWW2 and colony variants by stimulating reversion of the mucoid phenotype to the high pyocyanin expressing non-mucoid phenotype. The latter was highly virulent in the infection model with greater virulence when in co-culture with the streptococcus. The results of this study demonstrate that the oral commensal S. anginosus benefits from interaction with P. aeruginosa of the CF associated mucoid phenotype and modulates the behaviour of the pseudomonad in ways that may be clinically relevant.

Synergy between Colistin and the Signal Peptidase Inhibitor MD3 Is Dependent on the Mechanism of Colistin Resistance in Acinetobacter baumannii.

Synergy between colistin and the signal peptidase inhibitor MD3 was tested against isogenic mutants and clinical pairs of Acinetobacter baumannii isolates. Checkerboard assays and growth curves showed synergy against both colistin-susceptible strains (fractional inhibitory concentration index [FICindex] = 0.13 to 0.24) and colistin-resistant strains with mutations in pmrB and phosphoethanolamine modification of lipid A (FICindex = 0.14 to 0.25) but not against colistin-resistant Δlpx strains with loss of lipopolysaccharide (FICindex = 0.75 to 1). A colistin/MD3 combination would need to be targeted to strains with specific colistin resistance mechanisms.

Environment and colonisation sequence are key parameters driving cooperation and competition between Pseudomonas aeruginosa cystic fibrosis strains and oral commensal streptococci.

Cystic fibrosis (CF) patient airways harbour diverse microbial consortia that, in addition to the recognized principal pathogen Pseudomonas aeruginosa, include other bacteria commonly regarded as commensals. The latter include the oral (viridans) streptococci, which recent evidence indicates play an active role during infection of this environmentally diverse niche. As the interactions between inhabitants of the CF airway can potentially alter disease progression, it is important to identify key cooperators/competitors and environmental influences if therapeutic intervention is to be improved and pulmonary decline arrested. Importantly, we recently showed that virulence of the P. aeruginosa Liverpool Epidemic Strain (LES) could be potentiated by the Anginosus-group of streptococci (AGS). In the present study we explored the relationships between other viridans streptococci (Streptococcus oralis, Streptococcus mitis, Streptococcus gordonii and Streptococcus sanguinis) and the LES and observed that co-culture outcome was dependent upon inoculation sequence and environment. All four streptococcal species were shown to potentiate LES virulence factor production in co-culture biofilms. However, in the case of S. oralis interactions were environmentally determined; in air cooperation within a high cell density co-culture biofilm occurred together with stimulation of LES virulence factor production, while in an atmosphere containing added CO2 this species became a competitor antagonising LES growth through hydrogen peroxide (H2O2) production, significantly altering biofilm population dynamics and appearance. Streptococcus mitis, S. gordonii and S. sanguinis were also capable of H2O2 mediated inhibition of P. aeruginosa growth, but this was only visible when inoculated as a primary coloniser prior to introduction of the LES. Therefore, these observations, which are made in conditions relevant to the biology of CF disease pathogenesis, show that the pathogenic and colonisation potential of P. aeruginosa isolates can be modulated positively and negatively by the presence of oral commensal streptococci.

Activity of the type I signal peptidase inhibitor MD3 against multidrug-resistant Gram-negative bacteria alone and in combination with colistin.

OBJECTIVES: Effective treatment of Gram-negative bacterial infections is increasingly challenging due to the spread of multidrug-resistant strains and a lack of new antimicrobials in development. Bacterial type I signal peptidases (SPases) represent a highly conserved and essential target for inhibition by novel compounds. SPases are required for the effective processing of membrane translocated proteins involved in core functions related to metabolism, virulence and resistance. In this study we assessed the biochemical and functional activity of a novel synthetic inhibitor (MD3) of SPases against a wide range of Gram-negative pathogens. METHODS: The activity and specificity of MD3 for recombinant Pseudomonas aeruginosa SPase (LepB) and a genetically engineered LepB-regulatable strain were investigated. Antimicrobial activity of the compound alone and in combination with outer membrane-permeabilizing agents (sodium hexametaphosphate, colistin) was also determined against a collection of P. aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae and Stenotrophomonas maltophilia isolates. RESULTS: MD3 was found to inactivate the P. aeruginosa LepB protein (IC50 10 µM), resulting in antimicrobial effects potentiated in the presence of colistin. MD3 also demonstrated potent activity against wild-type and multidrug-resistant strains of A. baumannii and S. maltophilia with MICs ranging from 0.5 to 14 mg/L in the presence of subinhibitory concentrations of colistin. CONCLUSIONS: MD3 is a novel inhibitor of bacterial SPase in a range of non-fermentative Gram-negative bacteria. The antimicrobial activity is potentiated in combination with colistin and suggests that SPase inhibition warrants further exploration as a basis for future mono or combination therapies.

Differential potentiation of the virulence of the Pseudomonas aeruginosa cystic fibrosis liverpool epidemic strain by oral commensal Streptococci.

BACKGROUND: The Pseudomonas aeruginosa Liverpool epidemic strain (LES) is an important cystic fibrosis (CF) pathogen and is associated with increased morbidity and a worsened prognosis, compared with other CF-associated strains. However, interactions of common LES phenotypic variants with other members of the polymicrobial biofilms associated with chronic CF respiratory disease, such as oral commensal streptococci, have not been investigated. METHODS: Biofilm population dynamics, virulence factor production, and pathogenicity in Galleria mellonella larvae of common LES phenotypes (ie, low production, intermediate production, and overproduction of pyocyanin) in the presence or absence of anginosus group streptococci (AGS) were compared. RESULTS: AGS populations isolated from biofilm cocultures were P. aeruginosa phenotypic variant dependent, with higher AGS cell densities than those in monoculture frequently observed. Coexistence of AGS with a producer of low or intermediate levels of pyocyanin was found to result in enhancement of virulence factor production. In addition, the LES formed pathogenic partnerships with AGS in the G. mellonella infection model, with killing dependent on LES phenotype and AGS species. CONCLUSIONS: The pathogenic potential of LES phenotypic variants can be enhanced by the presence of oral commensal streptococci. As adaptive mutations leading to reduced virulence factor production are commonplace, the observations made are relevant in the general context of the biology of P. aeruginosa infection during CF.

Understanding and improving patient experience: a national survey of training courses provided by higher education providers and healthcare organizations in England.

BACKGROUND: Understanding and improving 'patient experience' is essential to delivering high quality healthcare. However, little is known about the provision of education and training to healthcare staff in this increasingly important area. OBJECTIVES: This study aims to ascertain the extent and nature of such provision in England and to identify how it might be developed in the future. METHODS: An on-line survey was designed to explore training provision relating to patient experiences. To ensure that respondents thought about patient experience in the same way we defined patient experience training as that which aims to teach staff: 'How to measure or monitor the experience, preferences and priorities of patients and use that knowledge to improve their experience'. Survey questions (n=15) were devised to cover nine consistently reported key aspects of patient experience; identified from the research literature and recommendations put forward by professional bodies. The survey was administered to (i) all 180 providers of Higher Education (HE) to student/qualified doctors, nurses and allied health professionals, and (ii) all 390 National Health Service (NHS) trusts in England. In addition, we added a single question to the NHS 2010 Staff Survey (n=306,000) relating to the training staff had received to deliver a good patient experience. RESULTS: Two hundred and sixty-five individuals responded to the on-line survey representing a total of 159 different organizations from the HE and healthcare sectors. Respondents most commonly identified 'relationships' as an 'essential' aspect of patient experience education and training. The biggest perceived gaps in current provision related to the 'physical' and 'measurement' aspects of our conceptualization of patient experience. Of the 148,657 staff who responded to the Staff Survey 41% said they had not received patient experience training and 22% said it was not applicable to them. CONCLUSIONS: While some relevant education courses are in place in England, the results suggest that specific training with regard to the physical needs and comfort of patients, and how patient experiences can be measured and used to improve services, should be introduced. Future developments should also focus, firstly, on involving a wider range of patients in planning and delivering courses and, secondly, evaluating whether courses impact on the attitudes and behaviors of different professional groups and might therefore contribute to improved patient experiences.

LacR mutations are frequently observed in Streptococcus intermedius and are responsible for increased intermedilysin production and virulence.

Streptococcus intermedius secretes a human-specific cytolysin, intermedilysin (ILY), which is considered to be the major virulence factor of this pathogen. We screened for a repressor of ily expression by using random gene disruption in a low-ILY-producing strain (PC574). Three independent high-ILY-producing colonies that had plasmid insertions within a gene that has high homology to lacR were isolated. Validation of these observations was achieved through disruption of lacR in strain PC574 with an erythromycin cassette, which also led to higher hemolytic activity, increased transcription of ily, and higher cytotoxicity against HepG2 cells, compared to the parental strain. The direct binding of LacR within the ily promoter region was shown by a biotinylated DNA probe pulldown assay, and the amount of ILY secreted into the culture supernatant by PC574 cells was increased by adding lactose or galactose to the medium as a carbon source. Furthermore, we examined lacR nucleotide sequences and the hemolytic activity of 50 strains isolated from clinical infections and 7 strains isolated from dental plaque. Of the 50 strains isolated from infections, 13 showed high ILY production, 11 of these 13 strains had one or more point mutations and/or an insertion mutation in LacR, and almost all mutations were associated with a marked decline in LacR function. These results strongly suggest that mutation in lacR is required for the overproduction of ILY, which is associated with an increase in pathogenicity of S. intermedius.

Pseudomonas aeruginosa possesses two putative type I signal peptidases, LepB and PA1303, each with distinct roles in physiology and virulence.

Type I signal peptidases (SPases) cleave signal peptides from proteins during translocation across biological membranes and hence play a vital role in cellular physiology. SPase activity is also of fundamental importance to the pathogenesis of infection for many bacteria, including Pseudomonas aeruginosa, which utilizes a variety of secreted virulence factors, such as proteases and toxins. P. aeruginosa possesses two noncontiguous SPase homologues, LepB (PA0768) and PA1303, which share 43% amino acid identity. Reverse transcription (RT)-PCR showed that both proteases were expressed, while a FRET-based assay using a peptide based on the signal sequence cleavage region of the secreted LasB elastase showed that recombinant LepB and PA1303 enzymes were both active. LepB is positioned within a genetic locus that resembles the locus containing the extensively characterized SPase of E. coli and is of similar size and topology. It was also shown to be essential for viability and to have high sequence identity with SPases from other pseudomonads (≥ 78%). In contrast, PA1303, which is small for a Gram-negative SPase (20 kDa), was found to be dispensable. Mutation of PA1303 resulted in an altered protein secretion profile and increased N-butanoyl homoserine lactone production and influenced several quorum-sensing-controlled phenotypic traits, including swarming motility and the production of rhamnolipid and elastinolytic activity. The data indicate different cellular roles for these P. aeruginosa SPase paralogues; the role of PA1303 is integrated with the quorum-sensing cascade and includes the suppression of virulence factor secretion and virulence-associated phenotypes, while LepB is the primary SPase.

A simple, semiselective medium for anaerobic isolation of anginosus group streptococci from patients with chronic lung disease.

The anaerobic isolation of anginosus group streptococci (AGS) from respiratory specimens containing diverse microbiota using a semiselective blood agar medium incorporating nalidixic acid and sulfamethazine (NAS) is described. AGS were detected in 60% of tested sputa from patients with cystic fibrosis, chronic obstructive pulmonary disease, and bronchiectasis. This demonstrates NAS to be a diagnostic tool for detecting AGS within the complex microbial communities associated with chronic lung disorders.

Comparative microarray analysis reveals that the core biofilm-associated transcriptome of Pseudomonas aeruginosa comprises relatively few genes.

Pseudomonas aeruginosa is a model organism for the study of intercellular communication and biofilm formation. As such, P. aeruginosa has been the subject of several microarray analyses comparing gene expression in biofilms and planktonic cultures. In the current work, we carried out a meta-analysis of these data sets to try and identify genes that are generically associated with biofilm formation in all of the conditions examined. Although the total number of transcripts modulated in the biofilms was large within the individual studies, the overlap between the data sets was small. Indeed, only five transcripts were upregulated and six transcripts were downregulated by more than twofold in the three data sets analysed. However, when the threshold modulation was relaxed to less than twofold, the overlap between the data sets increased, revealing a set of transcripts common to all of the studies. Transcriptional fusions and quantitative real-time PCR were used to independently confirm a selection of the observed modulations. Notably, we found that the expression profile of genes encoding the catabolic pathways for branched chain and aromatic amino acids was altered in biofilms, and that these alterations correlated with the onset of anaerobic growth. These findings were confirmed by quantitative amino acid analysis of culture supernatants. A mutant in one of the genes that we identified showed diminished biofilm formation in an attachment assay. The relatively small number of common biofilm-specific endpoint transcripts throws doubt on the suggestion that biofim formation proceeds through a pre-determined developmental pathway.

Pseudomonas aeruginosa PAO1 pyocin production affects population dynamics within mixed-culture biofilms.

Transcriptomic and phenotypic studies showed that pyocins are produced in Pseudomonas aeruginosa PAO1 aerobic and anaerobic biofilms. Pyocin activity was found to be high in slow-growing anaerobic biofilms but transient in aerobic biofilms. Biofilm coculture of strain PAO1 and a pyocin-sensitive isolate showed that pyocin production had a significant impact on bacterial population dynamics, particularly under anaerobic conditions.

Topographic distribution of bacteria associated with oral malodour on the tongue.

OBJECTIVE: To investigate the topographic distribution of bacterial types and loads associated with mid-morning oral malodour on the tongue surface. DESIGN: Fifty subjects with good oral health and at least 20 natural uncrowned teeth were included. Samples were taken with sterile brushes from the dorsal anterior (DA), dorsal middle (DM), dorsal posterior (DP), dorsal posterior to the circumvallate papillae (DPCP), lateral posterior (LP) and ventral posterior (VP) tongue surfaces. Samples were cultured on appropriate media for anaerobic bacteria, aerobic bacteria, Gram-negative anaerobic bacteria, volatile sulphur compound (VSC)-producing bacteria and Streptococcus saliuarius. Malodour was assessed by trained judges on an intensity basis. RESULTS: The counts of all bacterial groups were consistently highest at the DPCP surface. Mean VSC-producing bacterial counts (colony forming units/brush x10(5)) were 1.45, 5.67, 32.52, 88.94, 6.46 and 0.33 at DA, DM, DP, DPCP, LP and VP surfaces, respectively. Anaerobic, Gram-negative and VSC counts at DPCP surfaces increased with malodour intensity, whereas aerobic and S. saliuarius counts decreased; however these differences were not statistically significant. CONCLUSION: It is concluded that the DPCP area consistently carries the highest load of bacteria capable of contributing to oral malodour. The study demonstrates that tongue surfaces not accessible to routine oral hygiene procedures can significantly contribute to oral malodour.

Clustering of Pseudomonas aeruginosa transcriptomes from planktonic cultures, developing and mature biofilms reveals distinct expression profiles.

BACKGROUND: Pseudomonas aeruginosa is a genetically complex bacterium which can adopt and switch between a free-living or biofilm lifestyle, a versatility that enables it to thrive in many different environments and contributes to its success as a human pathogen. RESULTS: Transcriptomes derived from growth states relevant to the lifestyle of P. aeruginosa were clustered using three different methods (K-means, K-means spectral and hierarchical clustering). The culture conditions used for this study were; biofilms incubated for 8, 14, 24 and 48 hrs, and planktonic culture (logarithmic and stationary phase). This cluster analysis revealed the existence and provided a clear illustration of distinct expression profiles present in the dataset. Moreover, it gave an insight into which genes are up-regulated in planktonic, developing biofilm and confluent biofilm states. In addition, this analysis confirmed the contribution of quorum sensing (QS) and RpoS regulated genes to the biofilm mode of growth, and enabled the identification of a 60.69 Kbp region of the genome associated with stationary phase growth (stationary phase planktonic culture and confluent biofilms). CONCLUSION: This is the first study to use clustering to separate a large P. aeruginosa microarray dataset consisting of transcriptomes obtained from diverse conditions relevant to its growth, into different expression profiles. These distinct expression profiles not only reveal novel aspects of P. aeruginosa gene expression but also provide a growth specific transcriptomic reference dataset for the research community.

Transcriptome analysis of Pseudomonas aeruginosa growth: comparison of gene expression in planktonic cultures and developing and mature biofilms.

The transcriptomes of logarithmic- and stationary-phase Pseudomonas aeruginosa planktonic cultures and static biofilms of different stages of development were compared. Developing and confluent biofilm transcriptomes were found to be related to those of logarithmic- and stationary-phase planktonic cultures, respectively. In addition, a number of novel genes were up-regulated in developing and confluent biofilms, including genes encoding putative solute transport proteins and transcriptional regulators, respectively.

Spontaneous sequence duplications within capsule genes cap8E and tts control phase variation in Streptococcus pneumoniae serotypes 8 and 37.

Capsule phase variants were isolated from serotype 8 and serotype 37 pneumococcal sorbarods. Sequence duplications within the essential capsule genes - cap8E (type 8) and tts (type 37) - were found to introduce frameshifts and generate acapsular phenotypes. Capsular revertants possessed wild-type cap8E and tts genes, indicating the precise excision of these duplications. Reversion frequencies (OFF-ON) fit a linear relationship between log(frequency of reversion) and log(length of duplication), previously found for serotype three pneumococci [Waite, R. D., Struthers, J. K. & Dowson, C. G. (2001). Mol Microbiol 42, 1223-1232]. This study provides evidence that capsule phase variation can occur in pneumococcal serotypes with either simple (one to three genes) or complex capsule-encoding loci (12 genes). Given the key role of CapE (the first monosaccharide transferase) in other clinically important pneumococci, such as serotypes 14 and 19F with complex capsular loci, the observed duplication within cap8E suggests that capsule phase variation could be controlled by tandem sequence duplication in capE homologues in other pneumococcal serotypes that construct their capsules through polymerization of lipid-linked intermediates.