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Background: Analysis of " emerging " pathogens in cystic fibrosis (CF) lung disease has focused on unique pathogens that are rare in other human diseases or are drug resistant. Escherichia coli is recovered in the sputum of up to 25% of patients with CF, yet little is known about the epidemiology or clinical impact of infection., Methods: We studied patients attending a Canadian adult CF clinic who had positive sputum cultures for E coli from 1978 to 2016. Infection was categorized as transient or persistent (≥3 positive sputum cultures, spanning >6 months). Those with persistent infection were matched 2:1 with age, sex, and time-period controls without history of E coli infection, and mixed-effects models were used to assess pulmonary exacerbation (PEx) frequency, lung function decline, hospitalization, and intravenous antibiotic days., Results: Forty-five patients (12.3%) had E coli recovered from sputum samples between 1978 and 2016, and 18 patients (40%) developed persistent infection. Nine patients (24%) had PEx at incident infection, and increased bioburden was predictive of exacerbation ( P = .03). Risk factors for persistent infection included lower nutritional status ( P < .001) and lower lung function ( P = .009), but chronic infection with Pseudomonas aeruginosa was protective. There was no difference in annual lung function decline, need for hospitalization or intravenous antibiotics, or risk of PEx in patients with persistent infection., Conclusions: Persistent E coli infection was frequent and was more common in CF patients with low nutritional status and lung function. However, this does not predict clinical decline. Multicenter studies would allow better characterization of the epidemiology and clinical impact of E coli infection., (© The Author(s) 2019. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: To improve clinical outcomes, cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa infections are prescribed inhaled anti-pseudomonal antibiotics. Although, a diverse microbial community exists within CF airways, little is known about how the CF microbiota influences patient outcomes. We hypothesized that organisms within the CF microbiota are affected by inhaled-antibiotics and baseline microbiome may be used to predict therapeutic response., Methods: Adults with chronic P. aeruginosa infection from four clinics were observed during a single 28-day on/off inhaled-aztreonam cycle. Patients performed serial sputum collection, CF-respiratory infection symptom scores (CRISS), and spirometry. Patients achieving a decrease of ≥2 CRISS by day 28 were categorized as subjective responders (SR). The airway microbiome was defined by Illumina MiSeq analysis of the 16S rRNA gene., Results: Thirty-seven patients (median 37.4 years and FEV 1 44% predicted) were enrolled. No significant cohort-wide changes in the microbiome were observed between on/off AZLI cycles in either alpha- or beta-diversity metrics. However, at an individual level shifts were apparent. Twenty-one patients (57%) were SR and fourteen patients did not subjectively respond. While alpha-diversity metrics did not associate with response, patients who did not subjectively respond had a higher abundance of Staphylococcus and Streptococcus, and lower abundance of Haemophilus., Conclusions: The CF microbiome is relatively resilient to AZLI perturbations. However, associated changes were observed at the individual patient level. The relative abundance of key "off-target" organisms associated with subjective improvements suggesting that the microbiome may be used as a tool to predict patient response - potentially improving outcomes., (Copyright © 2019. Published by Elsevier B.V.)

A diverse microbiota exists within the airways of individuals with non-cystic fibrosis bronchiectasis (nCFB). How the lung microbiome evolves over time, and whether changes within the microbiome correlate with future disease progression is not yet known. We assessed the microbial community structure of 133 serial sputa and subsequent disease course of 29 nCFB patients collected over a span of 4-16 years using 16S rRNA paired-end sequencing. Interestingly, no significant shifts in the microbial community of individuals were observed during extended follow-up suggesting the microbiome remains relatively stable over prolonged periods. Samples that were Pseudomonas aeruginosa culture positive displayed markedly different microbial community structures compared to those that were positive for Haemophilus influenzae. Importantly, patients with sputum of lower microbial community diversity were more likely to experience subsequent lung function decline as defined by annual change in ≥-1 FEV 1 % predicted. Shannon diversity values <1 were more prevalent in patients with FEV 1 decline (P = 0.002). However, the relative abundance of particular core microbiota constituents did not associate with risk of decline. Here we present data confirming that the microbiome of nCFB individuals is generally stable, and that microbiome-based measurements may have a prognostic role as biomarkers for nCFB.

The natural history and epidemiology of Pseudomonas aeruginosa infections in non-cystic fibrosis (non-CF) bronchiectasis is not well understood. As such it was our intention to determine the evolution of airway infection and the transmission potential of P. aeruginosa in patients with non-CF bronchiectasis. A longitudinal cohort study was conducted from 1986-2011 using a biobank of prospectively collected isolates from patients with non-CF bronchiectasis. Patients included were ≥18 years old and had ≥2 positive P. aeruginosa cultures over a minimum 6-month period. All isolates obtained at first and most recent clinical encounters, as well as during exacerbations, that were morphologically distinct on MacConkey agar were genotyped by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 203 isolates from 39 patients were analysed. These were compared to a large collection of globally epidemic and local CF strains, as well as non-CF isolates. We identified four patterns of infection in non-CF bronchiectasis including: 1) persistence of a single strain (n=26; 67%); 2) strain displacement (n=8; 20%); 3) temporary disruption (n=3; 8%); and 4) chaotic airway infection (n=2; 5%). Patterns of infection were not significant predictors of rates of lung function decline or progression to end-stage disease and acquisition of new strains did not associate with the occurrence of exacerbations. Rarely, non-CF bronchiectasis strains with similar pulsotypes were observed in CF and non-CF controls, but no CF epidemic strains were observed. While rare shared strains were observed in non-CF bronchiectasis, whole-genome sequencing refuted patient-patient transmission. We observed a higher incidence of strain-displacement in our patient cohort compared to those observed in CF studies, although this did not impact on outcomes., Competing Interests: Conflict of interest: R. Somayaji has received research fellowship funding from Cystic Fibrosis Canada, Alberta Innovates and the Canadian Institutes of Health Research, and grants from Cystic Fibrosis Canada, the Canadian Institute of Health Research and the Royal College of Physicians and Surgeons outside the submitted work. M.D. Parkins has received research funding from Cystic Fibrosis Canada, the Canadian Institute of Health Research, the Lung Association of Alberta and the Northwest Territories, Gilead Sciences, The University of Calgary and Calgary Laboratory Services.

Biofilms composed of anaerobic bacteria can result in persistent infections and chronic inflammation. Host immune cells have difficulties clearing biofilm-related infections and this can result in tissue damage. Neutrophils are a vital component of the innate immune system and help clear biofilms. The comparative neutrophilic response to biofilms versus planktonic bacteria remains incompletely understood, particularly in the context of mixed infections. The objective of this study was to generate mixed species anaerobic bacterial biofilms composed of two opportunistic pathogens, Fusobacterium necrophorum and Porphyromonas levii, and evaluate neutrophil responses to extracellular fractions from both biofilms and planktonic cell co-cultures of the same bacteria. Purified bovine neutrophils exposed to culture supernatants from mixed species planktonic bacteria showed elevated oxidative activity compared to neutrophils exposed to biofilms composed of the same bacteria. Bacterial lipopolysaccharide plays a significant role in the stimulation of neutrophils; biofilms produced substantially more lipopolysaccharide than planktonic bacteria under these experimental conditions. Removal of lipopolysaccharide significantly reduced neutrophil oxidative response to culture supernatants of planktonic bacteria. Oxidative responses to LPS-removed biofilm supernatants and LPS-removed planktonic cell supernatants were similar. The limited neutrophil response to biofilm bacteria observed in this study supports the reduced ability of the innate immune system to eradicate biofilm-associated infections. Lipopolysaccharide is likely important in neutrophil response; however, the presence of other extracellular, immune modifying molecules in the bacterial media also appears to be important in altering neutrophil function., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Achromobacter species are increasingly being detected in cystic fibrosis (CF) patients, with an unclear epidemiology and impact. We studied a cohort of patients attending a Canadian adult CF clinic who had positive sputum cultures for Achromobacter species in the period from 1984 to 2013. Infection was categorized as transient or persistent (≥50% positive cultures for 1 year). Those with persistent infection were matched 2:1 with age-, sex-, and time-matched controls without a history of Achromobacter infection, and mixed-effects models were used to assess pulmonary exacerbation (PEx) frequency and lung function decline. Isolates from a biobank were retrospectively assessed, identified to the species level by nrdA sequencing, and genotyped using pulsed-field gel electrophoresis (PFGE). Thirty-four patients (11% of those in our clinic), with a median age of 24 years (interquartile range [IQR], 20.3 to 29.8 years), developed Achromobacter infection. Ten patients (29%) developed persistent infection. Persistence did not denote permanence, as most patients ultimately cleared infection, often after years. Patients were more likely to experience PEx at incident isolation than at prior or subsequent visits (odds ratio [OR], 2.7 [95% confidence interval {CI}, 1.2 to 6.7]; P = 0.03). Following persistent infection, there was no difference in annual lung function decline (-1.08% [95% CI, -2.73 to 0.57%] versus -2.74% [95% CI, -4.02 to 1.46%]; P = 0.12) or the odds of PEx (OR, 1.21 [95% CI, 0.45 to 3.28]; P = 0.70). Differential virulence among Achromobacter species was not observed, and no cases of transmission occurred. We demonstrated that incident Achromobacter infection was associated with a greater risk of PEx; however, neither transient nor chronic infection was associated with a worsened long-term prognosis. Large, multicenter studies are needed to clarify the clinical impact, natural history, and transmissibility of Achromobacter ., (Copyright © 2017 American Society for Microbiology.)

Background: Aztreonam lysine for inhalation (AZLI) is an inhaled antibiotic used to treat chronic Pseudomonas aeruginosa infection in CF. AZLI improves lung function and quality of life, and reduces exacerbations-improvements attributed to its antipseudomonal activity. Given the extremely high aztreonam concentrations achieved in the lower airways by nebulization, we speculate this may extend its spectrum of activity to other organisms. As such, we sought to determine if AZLI affects the CF lung microbiome and whether community constituents can be used to predict treatment responsiveness., Methods: Patients were included if they had chronic P. aeruginosa infection and repeated sputum samples collected before and after AZLI. Sputum DNA was extracted, and the V3-hypervariable region of the 16S ribosomal RNA (rRNA) gene amplified and sequenced., Results: Twenty-four patients naïve to AZLI contributed 162 samples. The cohort had a median age of 37.1 years, and a  median FEV 1 of 44% predicted. Fourteen patients were a priori defined as responders for achieving ≥3% FEV 1 improvement following initiation. No significant changes in alpha diversity were noted following AZLI. Furthermore, beta diversity demonstrated clustering with respect to patients, but had no association with AZLI use. However, we did observe a decline in the relative abundance of several individual operational taxonomic units (OTUs) following AZLI initiation suggesting that specific sub-populations of organisms may be impacted. Patients with higher abundance of Staphylococcus and anaerobic organisms including Prevotella and Fusobacterium were less likely to respond to therapy., Conclusions: Results from our study suggest potential alternate/additional mechanisms by which AZLI functions. Moreover, our study suggests that the CF microbiota may be used as a biomarker to predict patient responsiveness to therapy suggesting the microbiome may be harnessed for the personalization of therapies.

Pseudomonas aeruginosa develops profound population heterogeneity in CF airways. How changes in these populations relate to clinical status is unknown. In order to facilitate this understanding, frequent sampling of this community is required. To determine if the collection and storage of sputum at home may pose a viable option, we collected sputum from ten patients. Sputum samples were partitioned in two, with half immediately processed on MacConkey agar and half assessed after freezing for one week in a home-freezer. From each sample, 88 isolates were assessed for antibiotic susceptibility and virulence factor production. Freezing resulted in a 10 3 CFU/ml drop in P. aeruginosa. However, across 1760 isolates, no consistent difference in either antibiotic susceptibility nor virulence factors was observed suggesting freezing induced indiscriminate killing. Home collection and freezing of sputum will enable frequent and convenient assessment of P. aeruginosa population dynamics in CF., (Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

When cells mobilize nutrients from protein, they generate a fingerprint of peptide fragments that reflects the net action of proteases and the identities of the affected proteins. Analyzing these mixtures falls into a grey area between proteomics and metabolomics that is poorly served by existing technology. Herein, we describe an emerging digestomics strategy that bridges this gap and allows mixtures of proteolytic fragments to be quantitatively mapped with an amino acid level of resolution. We describe recent successes using this technique, including a case where digestomics provided the link between hemoglobin digestion by the malaria parasite and the world-wide distribution of chloroquine resistance. We highlight other areas of microbiology and cancer research that are well-suited to this emerging technology., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pseudomonas aeruginosa is a major pathogen in chronic lung diseases such as cystic fibrosis (CF) and non-cystic fibrosis bronchiectasis (nCFB). Much of our understanding regarding infections in nCFB patients is extrapolated from findings in CF with little direct investigation on the adaptation of P. aeruginosa in nCFB patients. As such, we investigated whether the adaptation of P. aeruginosa was indeed similar between nCFB and CF. From our prospectively collected biobank, we identified 40 nCFB patients who had repeated P. aeruginosa isolates separated by ≥6 months and compared these to a control population of 28 CF patients. A total of 84 nCFB isolates [40 early (defined as the earliest isolate in the biobank) and 41 late (defined as the last available isolate in the biobank)] were compared to 83 CF isolates (39 early and 44 late). We assessed the isolates for protease, lipase and elastase production; mucoid phenotype; swarm and swim motility; biofilm production; and the presence of the lasR mutant phenotype. Overall, we observed phenotypic heterogeneity in both nCFB and CF isolates and found that P. aeruginosa adapted to the nCFB lung environment similarly to the way observed in CF isolates in terms of protease and elastase expression, motility and biofilm formation. However, significant differences between nCFB and CF isolates were observed in lipase expression, which may allude to distinct characteristics found in the lung environment of nCFB patients. We also sought to determine virulence potential over time in nCFB P. aeruginosa isolates and found that virulence decreased over time, similar to CF.

Introduction: Cystic fibrosis (CF) airways are colonized by a polymicrobial community of organisms, termed the CF microbiota. We sought to define the microbial constituents of the home environment of individuals with CF and determine if it may serve as a latent reservoir for infection., Methods: Six patients with newly identified CF pathogens were included. An investigator collected repeat sputum and multiple environmental samples from their homes. Bacteria were cultured under both aerobic and anaerobic conditions. Morphologically distinct colonies were selected, purified and identified to the genus and species level through 16S rRNA gene sequencing. When concordant organisms were identified in sputum and environment, pulsed-field gel electrophoresis (PFGE) was performed to determine relatedness. Culture-independent bacterial profiling of each sample was carried out by Illumina sequencing of the V3 region of the 16s RNA gene., Results: New respiratory pathogens prompting investigation included: Mycobacterium abscessus(2), Stenotrophomonas maltophilia(3), Pseudomonas aeruginosa(3), Pseudomonas fluorescens(1), Nocardia spp.(1), and Achromobacter xylosoxidans(1). A median 25 organisms/patient were cultured from sputum. A median 125 organisms/home were cultured from environmental sites. Several organisms commonly found in the CF lung microbiome were identified within the home environments of these patients. Concordant species included members of the following genera: Brevibacterium(1), Microbacterium(1), Staphylococcus(3), Stenotrophomonas(2), Streptococcus(2), Sphingomonas(1), and Pseudomonas(4). PFGE confirmed related strains (one episode each of Sphinogomonas and P. aeruginosa) from the environment and airways were identified in two patients. Culture-independent assessment confirmed that many organisms were not identified using culture-dependent techniques., Conclusions: Members of the CF microbiota can be found as constituents of the home environment in individuals with CF. While the majority of isolates from the home environment were not genetically related to those isolated from the lower airways of individuals with CF suggesting alternate sources of infection were more common, a few genetically related isolates were indeed identified. As such, the home environment may rarely serve as either the source of infection or a persistent reservoir for re-infection after clearance.

The monitoring of epidemic Pseudomonas aeruginosa is important for cystic fibrosis (CF) infection control. The prairie epidemic strain (PES) is common in western Canadian CF clinics. Using whole-genome sequencing, we identified a novel genomic island and developed a PCR assay for PES. Against a collection of 186 P. aeruginosa isolates, the assay had 98% sensitivity and 100% specificity., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Epidemic strains of Pseudomonas aeruginosa have been found worldwide among the cystic fibrosis (CF) patient population. Using pulse-field gel electrophoresis, the Prairie Epidemic Strain (PES) has recently been found in one-third of patients attending the Calgary Adult CF Clinic in Canada. Using multi-locus sequence typing, PES isolates from unrelated patients were found to consistently have ST192. Though most patients acquired PES prior to enrolling in the clinic, some patients were observed to experience strain replacement upon transitioning to the clinic whereby local non-epidemic P. aeruginosa isolates were displaced by PES. Here we genotypically and phenotypically compared PES to other P. aeruginosa epidemic strains (OES) found around the world as well as local non-epidemic CF P. aeruginosa isolates in order to characterize PES. Since some epidemic strains are associated with worse clinical outcomes, we assessed the pathogenic potential of PES to determine if these isolates are virulent, shared properties with OES, and if its phenotypic properties may offer a competitive advantage in displacing local non-epidemic isolates during strain replacement. As such, we conducted a comparative analysis using fourteen phenotypic traits, including virulence factor production, biofilm formation, planktonic growth, mucoidy, and antibiotic susceptibility to characterize PES, OES, and local non-epidemic isolates. We observed that PES and OES could be differentiated from local non-epidemic isolates based on biofilm growth with PES isolates being more mucoid. Pairwise comparisons indicated that PES produced significantly higher levels of proteases and formed better biofilms than OES but were more susceptible to antibiotic treatment. Amongst five patients experiencing strain replacement, we found that super-infecting PES produced lower levels of proteases and elastases but were more resistant to antibiotics compared to the displaced non-epidemic isolates. This comparative analysis is the first to be completed on a large scale between groups of epidemic and non-epidemic CF P. aeruginosa isolates.

The Burkholderia cepacia complex (Bcc) is a collection of closely related, genetically distinct, ecologically diverse species known to cause life-threatening infections in cystic fibrosis (CF) patients. By virtue of a flexible genomic structure and diverse metabolic activity, Bcc bacteria employ a wide array of virulence factors for pathogenesis in CF patients and have developed resistance to most of the commonly used antibiotics. However, the mechanism of pathogenesis and antibiotic resistance is still not fully understood. This mini review discusses the established and potential virulence determinants of Bcc and some of the contemporary strategies including transcriptomics and proteomics used to identify these traits. We also propose the application of metabolic profiling, a cost-effective modern-day approach to achieve new insights.

Tritrpticin is an antimicrobial peptide with a strong microbicidal activity against Gram-positive and Gram-negative bacteria as well as fungi. The 13-residue peptide is essentially symmetrical and possesses a unique cluster of three Trp residues near the center of its amino acid sequence. The mechanism of action of tritrpticin is believed to involve permeabilization of the cytoplasmic membrane of susceptible bacteria. However it has been suggested that intracellular targets may also play a role in its antimicrobial activity. In this work the mechanism of action of several tritrpticin derivatives was studied through substitution of the three Trp residues with 5-hydroxy-tryptophan (5OHW), a naturally occurring non-ribosomal amino acid. Although it is more polar, 5OHW preserves many of the biophysical and biochemical properties of Trp, allowing the use of fluorescence spectroscopy and NMR techniques to study the interaction of the modified peptides with membrane mimetics. Single or triple 5OHW substitution did not have a large effect on the MIC of the parent peptide against Escherichia coli and Bacillus subtilis. However, the mechanism of action was altered by simultaneously replacing all three Trp with 5OHW. Our results suggest that the inner membrane of Gram-negative bacteria did not constitute the main target of this particular tritrpticin derivative. Since the addition of a hydroxyl group to the indole motif of the Trp residue was able to modify the mechanism of action of the peptides, our data confirm the importance of the Trp cluster in tritrpticin. This work also shows that 5OHW constitutes a new probe to modulate the antimicrobial activity and mechanism of action of other Trp-rich antimicrobial peptides., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Transmissible strains of Pseudomonas aeruginosa have been described for cystic fibrosis (CF) and may be associated with a worse prognosis. Using a comprehensive strain biobank spanning 3 decades, we sought to determine the prevalence and stability of chronic P. aeruginosa infection in an adult population. P. aeruginosa isolates from sputum samples collected at initial enrollment in our adult clinic and at the most recent clinic visit were examined by a combination of pulsed-field gel electrophoresis and multilocus sequence typing and compared against a collection of established transmissible and local non-CF bronchiectasis (nCFB) isolates. A total of 372 isolates from 107 patients, spanning 674 patient-years, including 66 patients with matched isolates from initial and final encounters, were screened. A novel clone with increased antibacterial resistance, termed the prairie epidemic strain (PES), was found in 29% (31/107 patients) of chronically infected patients referred from multiple prairie-based CF centers. This isolate was not found in those diagnosed with CF as adults or in a control population with nCFB. While 90% (60/66 patients) of patients had stable infection over a mean of 10.8 years, five patients experienced strain displacement of unique isolates, with PES occurring within 2 years of transitioning to adult care. PES has been present in our cohort since at least 1987, is unique to CF, generally establishes chronic infection during childhood, and has been found in patients at the time of transition of patients from multiple prairie-based CF clinics, suggesting broad endemicity. Studies are under way to evaluate the clinical implications of PES infection.

A tryptophan (Trp)-rich region in the wheat endosperm protein, puroindoline A, was previously shown to possess potent antimicrobial activity against Gram-positive and Gram-negative bacteria and this was attributed to the peptide inducing membrane instability. In the present work, the antimicrobial activity of the corresponding Trp-rich region in the puroindoline B isoform was examined and its antimicrobial activity was characterized. Unexpectedly, the puroindoline B Trp-rich peptide (PuroB) was relatively inactive compared to the related puroindoline A peptide (PuroA), despite strong sequence similarity. Using the sequence of PuroA as a template, a series of PuroB variants were synthesized and the antimicrobial activity was restored. Interestingly, all of these PuroB peptides preferentially interacted with negatively charged phospholipids, but unlike PuroA, they did not disrupt the integrity of lipid bilayers. This suggests that the primary mode of action of the PuroB peptides involves an antimicrobial target other than the bacterial membrane. Further tests revealed that all of the puroindoline derived peptides bind deoxyribonucleic acid (DNA) and block macromolecular synthesis in vivo. Based on these results, it appears that the interaction between puroindoline derived peptides and membranes is only an initial step in the mode of action and that binding to intracellular targets, such as DNA and ribonucleic acid (RNA), contributes significantly to their antimicrobial mode of action., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The virulence profiles of Pseudomonas aeruginosa quorum-sensing (QS) mutants were assessed in Drosophila melanogaster feeding and nicking infection models. Functional RhlIR and LasIR QS systems were required for killing in the fly feeding infection model but were not essential in the fly nicking infection model. Mixed infections between PAO1 and strains harbouring mutations in lasR, rhlI and lasI rhlI resulted in increased lethality in the fly feeding model compared with either isolate alone. These results suggested that the parental strain could cooperate with QS mutants in the Drosophila feeding infection model. Finally, the mixed infection between PAO1 and an rhlR mutant resulted in spiteful behaviour and reduced pathogenicity of the mixed culture.

The stringent response is a regulatory system that allows bacteria to sense and adapt to nutrient-poor environments. The central mediator of the stringent response is the molecule guanosine 3',5'-bispyrophosphate (ppGpp), which is synthesized by the enzymes RelA and SpoT and which is also degraded by SpoT. Our laboratory previously demonstrated that a relA mutant of Pseudomonas aeruginosa, the principal cause of lung infections in cystic fibrosis patients, was attenuated in virulence in a Drosophila melanogaster feeding model of infection. In this study, we examined the role of spoT in P. aeruginosa virulence. We generated an insertion mutation in spoT within the previously constructed relA mutant, thereby producing a ppGpp-devoid strain. The relA spoT double mutant was unable to establish a chronic infection in D. melanogaster and was also avirulent in the rat lung agar bead model of infection, a model in which the relA mutant is fully virulent. Synthesis of the virulence determinants pyocyanin, elastase, protease, and siderophores was impaired in the relA spoT double mutant. This mutant was also defective in swarming and twitching, but not in swimming motility. The relA spoT mutant and, to a lesser extent, the relA mutant were less able to withstand stresses such as heat shock and oxidative stress than the wild-type strain PAO1, which may partially account for the inability of the relA spoT mutant to successfully colonize the rat lung. Our results indicate that the stringent response, and SpoT in particular, is a crucial regulator of virulence processes in P. aeruginosa.

A number of human infections are characterized by the presence of more than one bacterial species and are defined as polymicrobial diseases. Methods for the analysis of the complex biological interactions in mixed infections with a large number of microorganisms are limited and do not effectively determine the contribution of each bacterial species to the pathogenesis of the polymicrobial community. We have developed a novel Drosophila melanogaster infection model to study microbe-microbe interactions and polymicrobe-host interactions. Using this infection model, we examined the interaction of 40 oropharyngeal isolates with Pseudomonas aeruginosa. We observe three classes of microorganisms, one of which acts synergistically with the principal pathogen, while being avirulent or even beneficial on its own. This synergy involves microbe-microbe interactions that result in the modulation of P. aeruginosa virulence factor gene expression within infected Drosophila. The host innate immune response to these natural-route polymicrobial infections is complex and characterized by additive, suppressive, and synergistic transcriptional activation of antimicrobial peptide genes. The polymicrobial infection model was used to differentiate the bacterial flora in cystic fibrosis (CF) sputum, revealing that a large proportion of the organisms in CF airways has the ability to influence the outcome of an infection when in combination with the principal CF pathogen P. aeruginosa.

Recently, two Drosophila melanogaster models of infection, fly feeding and fly nicking, have been developed that allow a determination of pathogenic potential of Pseudomonas aeruginosa isolates. In this study, control strains, isolates from burn wounds, and isolates from the sputa of cystic fibrosis (CF) patients were used to compare the two infection models to determine whether any of the isolates might be better adapted to either of the models. In addition, our goal was to determine the variability of isolates from individual CF patients. Three of four control strains (PAO1, PAK, and PA14) caused significant mortality in the flies in both models of infection. The remaining control strain, PA103, was lethal to flies in the nicking model but lacked significant lethality in the feeding model. The burn wound isolates had a high level of lethality in both models. Interestingly, the CF isolates had the largest diversity of lethality in both models of infection. The range of pathogenic potentials of the CF isolates occurred across a cohort of patients, both at the patient level and down to the level of individual sputum samples. The majority of all isolates had similar levels of lethality in both fly infection models. However, two CF isolates were significantly more lethal in the nicking model, and three CF isolates were significantly more lethal in the feeding model. In conclusion, the two Drosophila infection models were useful for the analysis of the diversity of pathogenic potentials of P. aeruginosa isolates.

The GacS/GacA two-component regulatory system in pseudomonads regulates genes involved in virulence, secondary metabolism and biofilm formation. Despite these regulatory functions, some Pseudomonas species are prone to spontaneous inactivating mutations in gacA and gacS. A gacS(-) strain of Pseudomonas aeruginosa PA14 was constructed to study the physiological role of this sensor histidine kinase. This loss-of-function mutation was associated with hypermotility, reduced production of acylhomoserine lactones, impaired biofilm maturation, and decreased antimicrobial resistance. Biofilms of the gacS(-) mutant gave rise to phenotypically stable small colony variants (SCVs) with increasing frequency when exposed to silver cations, hydrogen peroxide, human serum, or certain antibiotics (tobramicin, amikacin, azetronam, ceftrioxone, oxacilin, piperacillin or rifampicin). When cultured, the SCV produced thicker biofilms with greater cell density and greater antimicrobial resistance than did the wild-type or parental gacS(-) strains. Similar to other colony morphology variants described in the literature, this SCV was less motile than the wild-type strain and autoaggregated in broth culture. Complementation with gacS in trans restored the ability of the SCV to revert to a normal colony morphotype. These findings indicate that mutation of gacS is associated with the occurrence of stress-resistant SCV cells in P. aeruginosa biofilms and suggests that in some instances GacS may be necessary for reversion of these variants to a wild-type state.

Biofilm formation in Burkholderia cenocepacia has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the cepIR and cciIR quorum-sensing systems of Burkholderia cenocepacia K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the cepI and cepR mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the cciI mutant nor the double cciI cepI mutant was deficient in attachment, and restoration of the cciI gene resulted in less attachment than for the mutants. Meanwhile, the cciR mutant did show a significant reduction in attachment, as did the cciR cepIR mutant. While there was no change in antibiotic susceptibility with the individual cepIR and cciIR mutants, the cepI cciI mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the cepI and cepR mutants. Flow cell analysis of the individual cepIR mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of B. cenocepacia that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.

The stringent response is a mechanism by which bacteria adapt to nutritional deficiencies through the production of the guanine nucleotides ppGpp and pppGpp, produced by the RelA enzyme. We investigated the role of the relA gene in the ability of an extracellular pathogen, Pseudomonas aeruginosa, to cause infection. Strains lacking the relA gene were created from the prototypical laboratory strain PAO1 as well as the mucoid cystic fibrosis isolate 6106, which lacks functional quorum-sensing systems. The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. We found that strains lacking relA exhibited reduced virulence in a D. melanogaster feeding assay. In conditions of low magnesium, the relA gene enhanced production of the cell-cell signal N-[3-oxododecanoyl]-l-homoserine lactone, whereas relA reduced the production of the 2-heptyl-3-hydroxy-4-quinolone signal during serine hydroxamate induction of the stringent response. In the relA mutant, alterations in the Pseudomonas quinolone system pathways seemed to increase the production of pyocyanin and decrease the production of elastase. Deletion of relA also resulted in reduced levels of the RpoS sigma factor. These results suggest that adjustment of cellular ppGpp and pppGpp levels could be an important regulatory mechanism in P. aeruginosa adaptation in pathogenic relationships.

A colorimetric assay based on the reduction of a tetrazolium salt [2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT)] for rapidly determining the susceptibility of Pseudomonas aeruginosa isolates to bactericidal antibiotics is described. There was excellent agreement between the tobramycin and ofloxacin MICs determined after 5 h using the XTT assay and after 18 h using conventional methods. The data suggests that an XTT-based assay could provide a useful method for rapidly determining the susceptibility of P. aeruginosa to bactericidal antibiotics.

PvdS is an alternative sigma factor regulated by the global iron regulator Fur. It has been demonstrated that PvdS plays a role in the iron-dependent regulation of exotoxin A (ETA) in Pseudomonas aeruginosa strain PAO1. The goals of this research were to determine if pvdS was transcribed by the bacteria in the chronic lung infections associated with cystic fibrosis (CF) and to determine how PvdS interacts with the regAB promoters of the hyper-toxigenic strain PA103. It was found that pvdS is transcribed in the lungs of patients with CF and that it appears to be involved with the regulation of toxA in this environment. This correlated with the finding that in strain PA103, a mutation in pvdS reduced ETA activity while the same mutation in strain PAO1 abrogated ETA production. It was also shown that in strain PA103, pvdS was absolutely required for activation of the regAB P2 promoter. The effect of PvdS on the P2 promoter may be direct or indirect; however, in support of a direct role, an eight-out-of-nine base-pair match to the consensus sequence for PvdS binding was identified at the transcriptional start site for the P2 promoter. The effect of PvdS on the PA103 regAB P1 promoter under aerobic growth conditions was also examined. The results show that PvdS does modulate the expression from this promoter but that both the regAB operon and PvdS are required for optimal P1 promoter activity. These studies demonstrate that the alternative sigma factor PvdS acts as a regulator of ETA expression in P. aeruginosa strain PA103 through the regAB operon and that PvdS is expressed in lung infections associated with CF.

Individuals with cystic fibrosis (CF) are commonly colonized with Pseudomonas aeruginosa. The chronic infections caused by P. aeruginosa are punctuated by acute exacerbations of the lung disease, which lead to significant morbidity and mortality. As regulators of virulence determinants, P. aeruginosa quorum-sensing systems may be active in the chronic lung infections associated with CF. We have examined the levels of autoinducer molecules and transcript accumulation from the bacterial populations found in the lungs of patients with CF. We detected biologically active levels of N-(3-oxododecanoyl)-L-homoserine (3-oxo-C12-HSL) and N-butyryl-L-homoserine lactone (C4-HSL) in sputum from CF patients. Interestingly, it appears that C4-HSL is less frequently detected than 3-oxo-C12-HSL in the lungs of patients with CF. We also examined the transcription of the autoinducer synthase gene lasI and showed that it is frequently expressed in the lungs of patients with CF. We observed a significant correlation between the expression of lasI and four target genes of the Las quorum-sensing system. Taken together, our results indicate that quorum-sensing systems are active and may control virulence factor expression in the lungs of patients with CF.

We have investigated a potential role for GacA, the response regulator of the GacA/GacS two-component regulatory system, in Pseudomonas aeruginosa biofilm formation. When gacA was disrupted in strain PA14, a 10-fold reduction in biofilm formation capacity resulted relative to wild-type PA14. However, no significant difference was observed in the planktonic growth rate of PA14 gacA(-). Providing gacA in trans on the multicopy vector pUCP-gacA abrogated the biofilm formation defect. Scanning electron microscopy of biofilms formed by PA14 gacA(-) revealed diffuse clusters of cells that failed to aggregate into microcolonies, implying a deficit in biofilm development or surface translocation. Motility assays revealed no decrease in PA14 gacA(-) twitching or swimming abilities, indicating that the defect in biofilm formation is independent of flagellar-mediated attachment and solid surface translocation by pili. Autoinducer and alginate bioassays were performed similarly, and no difference in production levels was observed, indicating that this is not merely an upstream effect on either quorum sensing or alginate production. Antibiotic susceptibility profiling demonstrated that PA14 gacA(-) biofilms have moderately decreased resistance to a range of antibiotics relative to PA14 wild type. This study establishes GacA as a new and independent regulatory element in P. aeruginosa biofilm formation.

Pseudomonas aeruginosa biofilms are intrinsically resistant to antimicrobial chemotherapies. At present, very little is known about the physiological changes that occur during the transition from the planktonic to biofilm mode of growth. The resistance of P. aeruginosa biofilms to numerous antimicrobial agents that are substrates subject to active efflux from planktonic cells suggests that efflux pumps may substantially contribute to the innate resistance of biofilms. In this study, we investigated the expression of genes associated with two multidrug resistance (MDR) efflux pumps, MexAB-OprM and MexCD-OprJ, throughout the course of biofilm development. Using fusions to gfp, we were able to analyze spatial and temporal expression of mexA and mexC in the developing biofilm. Remarkably, expression of mexAB-oprM and mexCD-oprJ was not upregulated but rather decreased over time in the developing biofilm. Northern blot analysis confirmed that these pumps were not hyperexpressed in the biofilm. Furthermore, spatial differences in mexAB-oprM and mexCD-oprJ expression were observed, with maximal activity occurring at the biofilm substratum. Using a series of MDR mutants, we assessed the contribution of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps to P. aeruginosa biofilm resistance. These analyses led to the surprising discovery that the four characterized efflux pumps do not play a role in the antibiotic-resistant phenotype of P. aeruginosa biofilms.

Pseudomonas aeruginosa is an opportunistic human pathogen which poses a major threat to patients with cystic fibrosis (CF). Excessive amounts of mucus present in the lungs of CF patients promotes the colonization of P. aeruginosa. The migA gene, encoding a putative glycosyltransferase, has been shown to be highly inducible by respiratory mucus derived from CF patients. In this study, it is further demonstrated by population transcript analysis that the migA gene is highly expressed in the CF lung environment. Deletion analysis of the migA promoter identified a las-box-like sequence commonly found in promoters that are responsive to quorum sensing regulation. Further analysis of migA expression in quorum-sensing-defective strains, as well as its expression in response to autoinducer molecules, demonstrated that migA is regulated by the RhlI/RhlR quorum sensing regulatory system. Functionally, as the MigA sequence homology data suggested, the migA gene indeed affects the structure of LPS in P. aeruginosa. Increased expression of the migA gene results in a loss of core-plus-one LPS, while having no obvious effect on the long-chain O-antigen-bearing LPS. Although the exact biological role of the core-plus-one LPS is not clear, these experimental results suggest that migA up-regulation in the CF lung environment is part of the adaptive response which confers on P. aeruginosa a survival advantage.

The role of Pseudomonas aeruginosa exotoxin A (ETA) as a virulence factor in the lung infections of cystic fibrosis (CF) patients is not well understood. Transcript-accumulation studies of bacterial populations in sputum reveal high levels of transcription of toxA, which encodes ETA, in some patients with CF. However, in general, tissue damage in the lungs of patients with CF does not seem to be consistent with a high level of expression of active ETA. To address this discrepancy the authors analysed the production and activity of ETA produced by a number of P. aeruginosa CF isolates. One CF isolate, strain 4384, transcribed toxA at levels similar to the hypertoxigenic strain PA103 but produced an ETA with reduced ADP-ribosyltransferase (ADPRT) activity. Complementation in trans of strain 4384 with the wild-type toxA and a mixed toxin experiment suggested the absence of inhibitory accessory factors within this strain. The toxA gene from strain 4384 was cloned and sequenced, revealing only three mutations in the gene, all within the enzymic domain. The first mutation changed Ser-410 to Asn. The second mutation was located within an alpha-helix, altering Ala-476 to Glu. The third mutation, Ser-515 to Gly, was found at the protein surface. To date, Ser-410, Ala-476 and Ser-515 have not been reported to play a role in the ADPRT activity of ETA. However, it may be the combination of these mutations that reduces the enzymic activity of ETA produced by strain 4384. Expression of 4384 toxA and wild-type toxA in an isogenic strain revealed that 4384 ETA had 10-fold less ADPRT activity than wild-type ETA. ETA purified from strain 4384 also demonstrated 10-fold less ADPRT activity as compared to wild-type ETA. Cytotoxicity assays of purified ETA from strain 4384 indicated that the cytotoxicity of 4384 ETA is not reduced; it may be slightly more toxic than wild-type ETA. Analysis of five other CF isolates revealed a similar reduction in ADPRT activity to that seen in strain 4384. Sequence analysis of the enzymic domain of toxA from the five CF strains identified a number of mutations that could account for the reduction in ADPRT activity. These results suggest that some CF isolates produce an ETA with reduced enzymic activity and this may partially explain the pathogenesis of chronic lung infections of CF due to P. aeruginosa.

The exuberant immunoinflammatory response that is associated with Pseudomonas aeruginosa infection is the major source of the morbidity and mortality in cystic fibrosis (CF) patients. Previous studies have established that an exoproduct of P. aeruginosa (exoenzyme S) is a mitogen for human T lymphocytes and activates a larger percentage of T cells than most superantigens, which may contribute to the immunoinflammatory response. An animal model would facilitate studies of the pathophysiologic consequences of this activation. As a first step toward developing an animal model, the murine lymphocyte response to exoenzyme S was examined. When stimulated with exoenzyme S, splenocytes isolated from naive mice entered S phase and proliferated. The optimum response occurred after 2 to 3 days in culture, at 4 x 10(5) cells per well and 5.0 micrograms of exoenzyme S per ml. The response was not due to lipopolysaccharide, since Rhodobacter sphaeroides lipid A antagonist did not block the response. Other preparations of exoenzyme S stimulated lymphocyte proliferation, since the response to recombinant exoenzyme S (rHisExo S) cloned from strain 388 was similar to the response to exoenzyme S from strain DG1. There was evidence that genetic variability influenced the response, since A/J, CBA/J, and C57BL/6 mice were high responders and BALB/cJ mice were low responders following stimulation with exoenzyme S. Both splenic T and B lymphocytes entered the cell cycle in response to exoenzyme S. Thus, murine lymphocytes, like human lymphocytes, respond to P. aeruginosa exoenzyme S, which supports the development of a murine model that may facilitate our understanding of the role that exoenzyme S plays in the pathogenesis of P. aeruginosa infections in CF patients.

Exoenzyme S from P. aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 have distinct characteristics, which has led to a controversy about their homology and their pathophysiologic consequences. We have been investigating the ability of exoenzyme S to activate T lymphocytes, and therefore performed studies to determine whether exoenzyme S from P. aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 and expressed in Pseudomonas aeruginosa PA103 or in E. coli BL21(DE3), could induce T lymphocyte activation and proliferation. Both preparations were able to activate T cells and induce lymphocyte proliferation at similar levels as measured by flow cytometry of surface-activation markers and DNA synthesis, respectively. Further, a monoclonal antibody raised against exoenzyme S from strain DG1 partially neutralized T cell activation induced by recombinant exoenzyme S and bound to it in an immunoblot suggesting that the epitope responsible for T cell activation is shared by exoenzyme S from strain DG1 and recombinant exoenzyme S. These data suggest that the two different preparations of exoenzyme S, despite biochemical differences, share the characteristic that is responsible for T lymphocyte activation.

The role of Pseudomonas aeruginosa quorum-sensing systems in the lung infections associated with cystic fibrosis (CF) has not been examined. The purpose of this study was to determine if genes regulated by the LasR-LasI quorum-sensing system were coordinately regulated by the P. aeruginosa populations during the lung infections associated with CF. We also wanted to ascertain if there was a relationship between the expression of lasR, a transcriptional regulator, and some P. aeruginosa virulence factors during these infections. We extracted RNAs from the bacterial populations of 131 sputa taken from 23 CF patients. These RNAs were blotted and hybridized with probes to P. aeruginosa lasA, lasB, and toxA. The hybridization signals from each probe were ranked, and the rankings were analyzed by a Spearman rank correlation to determine if there was an association between the population transcript accumulations for the three genes. The correlations between the transcript accumulation patterns of pairs of the genes suggested that lasA, lasB, and toxA might be coordinately regulated during CF lung infections. To determine if this coordinate regulation might be due to regulation by LasR, we probed RNAs, extracted from 84 sputa, with the lasR, lasA, lasB, toxA, and algD probes. Statistical analysis indicated that lasR transcript accumulation correlated to lasA, lasB, toxA, and algD transcript accumulations. These results indicated that lasR may at least partially regulate or be coordinately regulated with lasA, lasB, toxA, and algD during the lung infections associated with CF. These results also suggested that the LasR-LasI quorum-sensing system may control the expression of at least some virulence factors in the lungs of patients with CF.

To investigate the regulatory role of the 5' untranslated region of Pseudomonas aeruginosa lasB mRNA, several lasB-lacZ protein and operon fusions were generated. By examining expression of these fusions in strain PAO1 we showed that the 5' untranslated leader sequence was involved in the post-transcriptional iron regulation of elastase. Two components of the unusually long 139 nucleotide lasB 5' untranslated region may be involved in post-transcriptional control. The first 101 nucleotides of the lasB mRNA may contain a translational enhancer element and an element that enhances transcript accumulation. However, the iron responsive element in the lasB mRNA requires sequences downstream of nucleotide +104.

Pseudomonas aeruginosa causes a chronic infection in the lungs of individuals with cystic fibrosis. The P. aeruginosa isolates from these infections, when grown under laboratory conditions, characteristically are mucoid and produce low levels of the more destructive virulence factors, such as exotoxin A and the proteases. We wanted to determine if during the chronic lung infections associated with CF, the expression of alginate was inversely correlated to the expression of exotoxin A, elastase, and the LasA protease. We measured the transcript accumulation of algD, a marker of alginate, toxA, the structural gene for exotoxin A, lasB, the structural gene for elastase, and lasA, the structural gene for LasA protease, from the sputum bacterial populations of 23 patients. In the 131 samples tested, we frequently detected transcripts from the four genes. When a Spearman rank correlation analysis was done on the samples, we found no correlation between algD transcript accumulation and toxA transcript accumulation. This result suggested that toxA was regulated independently of algD. Curiously, we found a positive correlation between algD transcript accumulation and both lasB and lasA transcript accumulation levels. This correlation may not indicate a direct association between algD and either lasA or lasB. More likely, it indicates a common regulatory element in a cascade of regulators or a common environmental cue that triggers transcription.

RegA is a transcriptional activator that controls exotoxin A (ETA) production in Pseudomonas aeruginosa. To date, functional assays performed with the purified protein have not clearly defined the molecular mechanism of action of RegA. In this study, we sought to identify important coding regions of regA by analysing the sequences around linker insertion mutations in regA that affected toxA transcription. First, we constructed a strain with the regAB locus deleted from the chromosome, PA103 delta regAB::Gm. toxA transcription was obliterated in strain PA103 delta regAB::Gm, demonstrating that the regAB locus is essential for ETA production. Next, we constructed a series of 6 bp linker insertion mutations distributed throughout regA. These regA linker insertion mutants were sequenced and screened in PA103 delta regAB::Gm for their effects on regulation of ETA production. Six linker insertion mutations occurring between amino acids (aa) 53 and 163 of RegA were isolated that resulted in depression of toxA transcription to varying levels relative to the parental regAB locus. One of these linker insertion mutations (pTR53), resulted in a lack of iron-regulated ETA production and occurred directly upstream from a predicted transmembrane alpha-helix. The other five linker mutations (pTR88, pTR124, pTR132, pTR132-2 and pTR163) occurred within or flanked a region of RegA between aa 87-142 with similarity to the transcriptional activation domains of ToxR, VirG and OmpR. These data suggest the presence of a previously unidentified transcriptional activation domain in RegA between aa 87-142 and implicate the predicted transmembrane alpha-helix in the N-terminus as being involved in sensory transduction.

In this study, we examined the regulation of exotoxin A (ETA) production by Pseudomonas aeruginosa during chronic lung infections of cystic fibrosis (CF) patients. We used a recently developed technique termed population transcript accumulation in hybridization studies with RNA extracted from sputa. With this technique, we demonstrated that the structural gene for ETA, toxA, as well as two genes encoding positive regulators of ETA synthesis, regA and regB, were expressed in the lungs of CF patients infected with P. aeruginosa. These genes were always expressed together, never alone or in pairs, suggesting coincident expression and a possible regulatory role for regA and regB in this environment. Fluctuations in the levels of the three gene products were observed among samples, consistent with a regulatory phenomenon. The level of regB RNA detected never exceeded that of regA, although the ratio of regA RNA to regB RNA detected did change between samples. These observations are in agreement with in vitro observations which have shown that regB is located 3' to regA in an operon which is expressed from two independently regulated promoters located upstream of regA. The presence of high levels of toxA, regA, and regB RNAs in some sputum samples prompted us to look for hyperproducing-toxin strains in the sputa of CF patients. In vitro, one such strain, 4384, had a transcript accumulation pattern for toxA, regA, and regB similar to that of a laboratory hyperproducer of ETA, strain PA103. These observations suggest that regA and regB are involved in the regulation of ETA production in strains of P. aeruginosa infecting the lungs of CF patients and that some of these strains may regulate ETA production in a manner similar to that of the hyperproducing-ETA strain PA103.

The conversion of Pseudomonas aeruginosa PAO to the mucoid phenotype has been reported for a chronic pulmonary infection model in rats (D. E. Woods, P. A. Sokol, L. E. Bryan, D. G. Storey, S. J. Mattingly, H. J. Vogel, and H. Ceri, J. Infect. Dis. 163:143-149, 1991). This conversion was associated with a genetic rearrangement upstream of the exotoxin A gene. To characterize the genetic rearrangement, the region upstream of the toxA gene was cloned from PAO, PAO-muc (a mucoid strain), and PAO-rev (a nonmucoid revertant strain). The nucleotide sequence of a 4.8-kb fragment from PAO-muc was determined. A+T-rich regions of approximately 2 kb (IS-PA-4) and 0.4 kb (IS-PA-5) were identified in this fragment. DNA probes constructed internal to these regions hybridized to PAO-muc but not to PAO or PAO-rev, suggesting that PAO-muc contains an insertion element. Sequence analysis of the nonmucoid clones indicated that a 2,561-bp fragment corresponding to IS-PA-4 and a 992-bp fragment corresponding to IS-PA-5 were not present in PAO or PAO-rev. Both nonmucoid clones, however, contained in the same location as IS-PA-4, a 1,313-bp region which was not present in PAO-muc. DNA probes complementary to this sequence, designated IS-PA-6, did not hybridize with PAO-muc, indicating that this sequence had been replaced upon conversion to the mucoid phenotype. Between IS-PA-4 and IS-PA-5 there was a 500-bp sequence which was 94% identical to the 500-bp sequence downstream of IS-PA-6. These insertion elements had some DNA sequence similarity to plasmid and transposon sequences, suggesting that they may be of plasmid origin. IS-PA-4 and IS-PA-5 were shown also to be present in two mucoid isolates from cystic fibrosis patients. The insertions occurred in the same location upstream of the toxA gene, suggesting that this type of genetic recombination may also be associated with mucoid conversion in some P. aeruginosa clinical isolates.

The in vivo regulation of Pseudomonas aeruginosa virulence factors during the chronic lung infections associated with cystic fibrosis is poorly understood. We have developed an approach for the analysis of transcript accumulation of individual virulence factors from the P. aeruginosa populations found in the sputa of patients with cystic fibrosis. This method has been named population transcript accumulation, since we examine the transcript accumulation patterns in RNA extracted from the total bacterial population found in the sputum samples. DNA probes specific for P. aeruginosa elastase (lasB) and exotoxin A (toxA) were used to examine the population transcript accumulation of 21 sputum samples taken from 10 patients. We detected three patterns of population transcript accumulation: lasB and toxA, lasB alone, and neither lasB nor toxA. We also measured the relative levels of elastase and exotoxin A transcript accumulation in 19 of these samples. In the six samples containing both toxA and lasB transcripts, we found that the levels of lasB transcripts were consistently higher than those of toxA. Differences in the stability of the two mRNA species could not completely account for the higher level of lasB population transcript accumulation, since we showed that the mRNA half-life of lasB (11 min) was similar to that of toxA (10 min). Finally, we showed that elastase transcripts could be detected in some samples which contained only mucoid isolates. This finding suggests that both mucoid and nonmucoid populations may be transcribing lasB in the lungs of patients with cystic fibrosis.

A lasB-lacZ translational fusion (pTS400) was used to examine expression of the elastase gene (lasB) in Pseudomonas aeruginosa strain PAO1. Expression from the lasB-lacZ fusion was enhanced when PAO1(pTS400) was grown in a defined medium containing elevated levels of zinc (6.0 micrograms ml-1). Transcript accumulation studies on PAO1(pTS400) and PAO1 showed that the addition of zinc had a slight negative effect on lasB transcription. These results indicated that zinc regulates the expression of elastase at the translational level. A comparison between zinc regulation and iron regulation was also made. Iron has a negative effect on lasB-lacZ expression. When PAO1(pTS400) was grown in a defined medium with a low iron content (0.1 microgram ml-1) the bacteria still responded to zinc. The independent effects of low iron and high zinc concentrations suggest separate control mechanisms for the two factors. Transcript accumulation studies on PAO1 and PAO1 (pTS400) indicated that early in the growth curve iron did not influence transcription of lasB or lasB-lacZ. Later in the growth curve a slight increase in lasB-lacZ transcription was observed only in PAO1(pTS400) grown in low iron. These results suggest that the iron regulation of lasB occurs predominantly at the translational level. Finally, when PAO1(pTS400) was grown in a complex peptone-based medium, a high level of transcript accumulation accounted for elastase expression. Alterations of iron and zinc concentrations of this medium did not affect the expression of elastase. These results suggest that there may be additional environmental cues regulating lasB transcription.

A multicopy plasmid containing the Escherichia coli fur gene was introduced into Pseudomonas aeruginosa strain PA103C. This strain contains a toxA-lacZ fusion integrated into its chromosome at the toxA locus. Beta-galactosidase synthesis in this strain is regulated by iron, as is seen for exotoxin A production. Beta-galactosidase synthesis and exotoxin A production in PA103C containing multiple copies of E. coli fur was still repressed in low iron conditions. The transcription of regA, a positive regulator of toxA, was also found to be inhibited by multiple copies of the E. coli fur gene. In addition, the ability of PA103C containing multiple copies of E. coli fur to produce protease was greatly reduced relative to PA103C containing a vector control. A polyclonal rabbit serum containing antibodies that recognize E. coli Fur was used to screen whole-cell extracts from Vibrio cholerae, Shigella flexneri, Salmonella typhimurium and Pseudomonas aeruginosa. All strains tested expressed a protein that was specifically recognized by the anti-Fur serum. These results and those described above suggest that Fur structure and function are conserved in a variety of distinct bacterial genera and that at least some of these different genera use this regulatory protein to control genes encoding virulence factors.

Exotoxin A production in Pseudomonas aeruginosa is dependent on two regulatory genes, regA and regB, which are located in tandem on the chromosome. Expression of regA and regB is controlled by two promoters (P1 and P2) situated upstream of the regAB locus. We have studied the effect of the regA and regB gene products on transcription from the regAB promoters. Transcriptional and translational fusions, under the control of the P. aeruginosa regA promoters, were used to analyze the regulation of these promoters in a variety of genetic backgrounds. When the regA P1 promoter was supplied in trans to strains lacking expression of regB (PAO1) or lacking transcription of the regAB operon (PA103-29), little activity from the P1 promoter was detected. In contrast, activity from the P2 promoter was not affected in either PAO1 or PA103-29. Sequence analysis of the regAB operon of PA103-29 detected two mutations. One of the mutations is predicted to result in a premature stop codon in the regA open reading frame. We complemented PA103-29 with a construction containing regA and an inactive regB or a construction containing both regA and regB to directly analyze the effect of regB on transcription of the regAB operon. When PA103-29 was complemented with regA but not regB, we could not detect any transcription from the P1 promoter. Complementation of PA103-29 with both regA and regB resulted in a high level of transcription from the P1 promoter and a corresponding early transcriptional activation of toxA. Our results indicated that induction of transcription from the P1 promoter requires the regB open reading frame and thus the regAB operon is autogenously regulated in P. aeruginosa.

A chronic pulmonary infection model was used to induce conversion to the mucoid phenotype by Pseudomonas aeruginosa PAO. At 6 months after initial inoculation, organisms isolated from infected lungs demonstrated the mucoid phenotype. Significant decreases (P less than .01) were seen in the levels of exotoxin A, exoenzyme S, phospholipase C, and pyochelin produced by the mucoid P. aeruginosa PAO rat lung isolates that returned to parental levels after reversion to the nonmucoid phenotype. In addition, lipopolysaccharide of the mucoid PAO lung isolates failed to react with serotype B-specific antibody in contrast to the original PAO and the revertant PAO organisms. Digestion of chromosomal DNA and hybridization with P. aeruginosa virulence factor-specific probes demonstrated that conversion to the mucoid phenotype was associated with rearrangement of chromosomal DNA upstream of the exotoxin A gene. Analysis of DNA from revertant organisms revealed hybridization patterns identical to the original PAO organism.

The yield of exotoxin A from Pseudomonas aeruginosa has been shown to be strain-dependent. Exotoxin A production requires the presence of the positive regulatory gene, regA. We cloned the regA genetic locus from the prototypical P. aeruginosa strain PAO1 and examined its ability to influence exotoxin A yields compared to the same region cloned from the hypertoxin-producing strain, PA103. The P. aeruginosa regA mutant strain, PA103-29, containing the PAO1 regA locus in trans produced approximately five to seven times less extracellular exotoxin A than PA103-29 containing the regA locus cloned from the hypertoxigenic strain, PA103. Nucleotide sequence analysis of the PAO1 regA locus revealed several differences, the most striking of which was the absence of a second open reading frame that was present in the analogous PA103 DNA. In addition, an amino acid substitution was found at position 144 of RegA (Thr in PAO1 and Ala in PA103). Recombinant molecules were constructed to test the contribution of each of these changes in nucleotide sequence on extracellular exotoxin A yields. The amino acid substitution in the PAO1 RegA protein was found not to affect overall exotoxin A yields. In contrast, the presence of the second open reading frame immediately downstream of the PA103 regA gene was found to influence extracellular exotoxin A yields. This open reading frame encodes a gene which we call regB. Nucleotide sequence analysis indicates that regB is 228 nucleotides in length and encodes a protein of 7527 Daltons. Our data suggest that regB is required for optimal exotoxin A production and its absence in strain PAO1 partially accounts for the difference in yield of extracellular exotoxin A between P. aeruginosa strains PAO1 and PA103.

Expression studies utilizing the regA promoters, fused in tandem or separately to promoterless reporter genes, indicated that regA is transcribed from two promoters (P1 and P2). Both promoters can act independently. Expression from the P1 promoter is not affected by the iron content of the medium. Expression from the P2 promoter is tightly regulated by iron.

Iron regulation of toxA and regA transcript accumulation was examined in Pseudomonas aeruginosa PA103 containing the regA gene on a multicopy plasmid. The patterns of transcript accumulation for toxA and regA were found to be positively correlated. Dot blot and Northern (RNA) blot analysis of total RNA isolated throughout the bacterial growth cycle indicated that multiple copies of the regA gene uncoupled iron repression of the first phase of transcript accumulation for both regA and toxA genes. However, regulation by iron of the second phase of transcript accumulation for each gene was unaffected by several regA gene copies. Total toxin production was increased in cells with multiple copies of regA grown in either low- or high-iron medium. Primer extension analysis of regA mRNA extracted from cells grown in high- and low-iron medium and examined at different points in the cell growth cycle supported the hypothesis that iron regulation of regA transcription occurs at the level of transcriptional initiation. Two start sites were shown for regA transcription at -164 and -75 base pairs from the ATG start codon. The differential regulation of regA transcript accumulation when regA is present in single or multiple copy and the mapping of independent start sites for regA mRNA support the evidence that regA transcription is directed by independently regulated promoter regions.