Richard D. Waite

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.

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

BackgroundPseudomonas 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.ResultsTranscriptomes 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).ConclusionThis 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.

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.

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.

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.

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.

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.

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

ABSTRACT 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.

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.