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Background: Antimicrobial resistance (AMR) is an intensifying threat that requires urgent mitigation to avoid a post-antibiotic era. Pseudomonas aeruginosa represents one of the greatest AMR concerns due to increasing multi- and pan-drug resistance rates. Shotgun sequencing is gaining traction for in silico AMR profiling due to its unambiguity and transferability; however, accurate and comprehensive AMR prediction from P. aeruginosa genomes remains an unsolved problem., Methods: We first curated the most comprehensive database yet of known P. aeruginosa AMR variants. Next, we performed comparative genomics and microbial genome-wide association study analysis across a Global isolate Dataset (n = 1877) with paired antimicrobial phenotype and genomic data to identify novel AMR variants. Finally, the performance of our P. aeruginosa AMR database, implemented in our AMR detection and prediction tool, ARDaP, was compared with three previously published in silico AMR gene detection or phenotype prediction tools-abritAMR, AMRFinderPlus, ResFinder-across both the Global Dataset and an analysis-naïve Validation Dataset (n = 102)., Results: Our AMR database comprises 3639 mobile AMR genes and 728 chromosomal variants, including 75 previously unreported chromosomal AMR variants, 10 variants associated with unusual antimicrobial susceptibility, and 281 chromosomal variants that we show are unlikely to confer AMR. Our pipeline achieved a genotype-phenotype balanced accuracy (bACC) of 85% and 81% across 10 clinically relevant antibiotics when tested against the Global and Validation Datasets, respectively, vs. just 56% and 54% with abritAMR, 58% and 54% with AMRFinderPlus, and 60% and 53% with ResFinder. ARDaP's superior performance was predominantly due to the inclusion of chromosomal AMR variants, which are generally not identified with most AMR identification tools., Conclusions: Our ARDaP software and associated AMR variant database provides an accurate tool for predicting AMR phenotypes in P. aeruginosa, far surpassing the performance of current tools. Implementation of ARDaP for routine AMR prediction from P. aeruginosa genomes and metagenomes will improve AMR identification, addressing a critical facet in combatting this treatment-refractory pathogen. However, knowledge gaps remain in our understanding of the P. aeruginosa resistome, particularly the basis of colistin AMR., (© 2024. The Author(s).)

Pseudomonas aeruginosa ( Pa ) is the predominant bacterial pathogen in people with cystic fibrosis (CF) and can be transmitted by airborne droplet nuclei. Little is known about the ability of ultraviolet band C (UV-C) irradiation to inactivate Pa at doses and conditions relevant to implementation in indoor clinical settings. We assessed the effectiveness of UV-C (265 nm) at up to seven doses on the decay of nebulized Pa aerosols (clonal Pa strain) under a range of experimental conditions. Experiments were done in a 400 L rotating sampling drum. A six-stage Andersen cascade impactor was used to collect aerosols inside the drum and the particle size distribution was characterized by an optical particle counter. UV-C effectiveness was characterized relative to control tests (no UV-C) of the natural decay of Pa . We performed 112 tests in total across all experimental conditions. The addition of UV-C significantly increased the inactivation of Pa compared with natural decay alone at all but one of the UV-C doses assessed. UV-C doses from 246-1968 µW s/cm 2 had an estimated effectiveness of approximately 50-90% for airborne Pa . The effectiveness of doses ≥984 µW s/cm 2 were not significantly different from each other ( p -values: 0.365 to ~1), consistent with a flattening of effectiveness at higher doses. Modelling showed that delivering the highest dose associated with significant improvement in effectiveness (984 µW s/cm 2 ) to the upper air of three clinical rooms would lead to lower room doses from 37-49% of the 8 h occupational limit. Our results suggest that UV-C can expedite the inactivation of nebulized airborne Pa under controlled conditions, at levels that can be delivered safely in occupied settings. These findings need corroboration, but UV-C may have potential applications in locations where people with CF congregate, coupled with other indoor and administrative infection control measures.

Background. Antimicrobial resistance (AMR) is an ever-increasing global health concern. One crucial facet in tackling the AMR epidemic is earlier and more accurate AMR diagnosis, particularly in the dangerous and highly multi-drug-resistant ESKAPE pathogen, Pseudomonas aeruginosa . Objectives. We aimed to develop two SYBR Green-based mismatch amplification mutation assays (SYBR-MAMAs) targeting GyrA T83I ( gyrA 248) and GyrA D87N, D87Y and D87H ( gyrA 259). Together, these variants cause the majority of fluoroquinolone (FQ) AMR in P. aeruginosa . Methods. Following assay validation, the gyrA 248 and gyrA 259 SYBR-MAMAs were tested on 84 Australian clinical P. aeruginosa isolates, 46 of which demonstrated intermediate/full ciprofloxacin resistance according to antimicrobial susceptibility testing. Results. Our two SYBR-MAMAs correctly predicted an AMR phenotype in the majority (83%) of isolates with intermediate/full FQ resistance. All FQ-sensitive strains were predicted to have a sensitive phenotype. Whole-genome sequencing confirmed 100 % concordance with SYBR-MAMA genotypes. Conclusions. Our GyrA SYBR-MAMAs provide a rapid and cost-effective method for same-day identification of FQ AMR in P. aeruginosa . An additional SYBR-MAMA targeting the GyrB S466Y/S466F variants would increase FQ AMR prediction to 91 %. Clinical implementation of our assays will permit more timely treatment alterations in cases where decreased FQ susceptibility is identified, leading to improved patient outcomes and antimicrobial stewardship.

Pseudomonas aeruginosa is one of the principal pathogens implicated in respiratory infections of patients with cystic fibrosis (CF) and non-CF bronchiectasis. Previously, we demonstrated that impaired serum-mediated killing of P. aeruginosa was associated with increased severity of respiratory infections in patients with non-CF bronchiectasis. This inhibition was mediated by high titers of O-antigen-specific IgG2 antibodies that cloak the surface of the bacteria, blocking access to the membrane. Infection-related symptomatology was ameliorated in patients by using plasmapheresis to remove the offending antibodies. To determine if these inhibitory "cloaking antibodies" were prevalent in patients with CF, we investigated 70 serum samples from patients with P. aeruginosa infection and 5 from those without P. aeruginosa infection. Of these patients, 32% had serum that inhibited the ability of healthy control serum to kill P. aeruginosa. Here, we demonstrate that this inhibition of killing requires O-antigen expression. Furthermore, we reveal that while IgG alone can inhibit the activity of healthy control serum, O-antigen-specific IgA in patient sera can also inhibit serum-killing. We found that antibody affinity, not just titer, was also important in the inhibition of serum-mediated killing. These studies provide novel insight into cloaking antibodies in human infection and may provide further options in CF and other diseases for treatment of recalcitrant P. aeruginosa infections.

Patient-derived isolates of the opportunistic pathogen Pseudomonas aeruginosa are frequently resistant to antibiotics due to the presence of sequence variants in resistance-associated genes. However, the frequency of antibiotic resistance and of resistance-associated sequence variants in environmental isolates of P. aeruginosa has not been well studied. Antimicrobial susceptibility testing (ciprofloxacin, ceftazidime, meropenem, tobramycin) of environmental ( n =50) and cystic fibrosis ( n =42) P. aeruginosa isolates was carried out. Following whole genome sequencing of all isolates, 25 resistance-associated genes were analysed for the presence of likely function-altering sequence variants. Environmental isolates were susceptible to all antibiotics with one exception, whereas patient-derived isolates had significant frequencies of resistance to each antibiotic and a greater number of likely resistance-associated genetic variants. These findings indicate that the natural environment does not act as a reservoir of antibiotic-resistant P. aeruginosa, supporting a model in which antibiotic susceptible environmental bacteria infect patients and develop resistance during infection.

Purpose of Review: This review provides an update on definitions of chronicity of infection, approaches to airway sampling to detect infection, strategies for Pseudomonas aeruginosa eradication, impact of cystic fibrosis transmembrane regulator protein (CFTR) modulators and future challenges for clinical trials., Recent Findings: Rates of P. aeruginosa have decreased over the past two decades with establishment of effective eradication protocols. Definitions of chronic P. aeruginosa infection have required adaptation for healthier populations. Although molecular (PCR) approaches to early P. aeruginosa detection are sensitive, to date, earlier diagnosis has not impacted on clinical outcomes. Despite eradication regimens, some people with early P. aeruginosa fail to clear their infection. Most people also experience a recurrence and eventual transition to chronic infection. Several recent studies sought to address this gap. CFTR modulators (predominantly ivacaftor) demonstrated reduced P. aeruginosa density, although infection may persist or recur demonstrating the need for continued antiinfective therapies in the modulator era., Summary: Future studies of approaches to P. aeruginosa eradication will be complex due to expanded availability and ongoing competitive clinical trials of CFTR modulators. Studies to address optimal eradication therapy, particularly in adults, will be required, though adequate recruitment to power these studies may prove challenging.

Background and Objective: Aerosol transmission of Pseudomonas aeruginosa has been suggested as a possible mode of respiratory infection spread in patients with cystic fibrosis (CF); however, whether this occurs in other suppurative lung diseases is unknown. Therefore, we aimed to determine if (i) patients with bronchiectasis (unrelated to CF) or chronic obstructive pulmonary disease (COPD) can aerosolize P. aeruginosa during coughing and (ii) if genetically indistinguishable (shared) P. aeruginosa strains are present in these disease cohorts., Methods: People with bronchiectasis or COPD and P. aeruginosa respiratory infection were recruited for two studies. Aerosol study: Participants (n = 20) underwent cough testing using validated cough rigs to determine the survival of P. aeruginosa aerosols in the air over distance and duration. Genotyping study: P. aeruginosa sputum isolates (n = 95) were genotyped using the iPLEX20SNP platform, with a subset subjected to the enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) assay to ascertain their genetic relatedness., Results: Aerosol study: Overall, 7 of 20 (35%) participants released P. aeruginosa cough aerosols during at least one of the cough aerosol tests. These cough aerosols remained viable for 4 m from the source and for 15 min after coughing. The mean total aerosol count of P. aeruginosa at 2 m was two colony-forming units. Typing study: No shared P. aeruginosa strains were identified., Conclusion: Low viable count of P. aeruginosa cough aerosols and a lack of shared P. aeruginosa strains observed suggest that aerosol transmission of P. aeruginosa is an unlikely mode of respiratory infection spread in patients with bronchiectasis and COPD., (© 2019 Asian Pacific Society of Respirology.)

The lungs of individuals with cystic fibrosis (CF) become chronically infected with Pseudomonas aeruginosa that is difficult to eradicate by antibiotic treatment. Two key P. aeruginosa antibiotic resistance mechanisms are the AmpC β-lactamase that degrades β-lactam antibiotics and MexXYOprM, a three-protein efflux pump that expels aminoglycoside antibiotics from the bacterial cells. Levels of antibiotic resistance gene expression are likely to be a key factor in antibiotic resistance but have not been determined during infection. The aims of this research were to investigate the expression of the ampC and mexX genes during infection in patients with CF and in bacteria isolated from the same patients and grown under laboratory conditions. P. aeruginosa isolates from 36 CF patients were grown in laboratory culture and gene expression measured by reverse transcription-quantitative PCR (RT-qPCR). The expression of ampC varied over 20,000-fold and that of mexX over 2,000-fold between isolates. The median expression levels of both genes were increased by the presence of subinhibitory concentrations of antibiotics. To measure P. aeruginosa gene expression during infection, we carried out RT-qPCR using RNA extracted from fresh sputum samples obtained from 31 patients. The expression of ampC varied over 4,000-fold, while mexX expression varied over 100-fold, between patients. Despite these wide variations, median levels of expression of ampC in bacteria in sputum were similar to those in laboratory-grown bacteria. The expression of mexX was higher in sputum than in laboratory-grown bacteria. Overall, our data demonstrate that genes that contribute to antibiotic resistance can be highly expressed in patients, but there is extensive isolate-to-isolate and patient-to-patient variation., (Copyright © 2018 American Society for Microbiology.)

Background: Chronic lung infections caused by Pseudomonas aeruginosa are a significant cause of morbidity and mortality in people with cystic fibrosis (CF). Shared P. aeruginosa strains, that can be transmitted between patients, are of concern and in Australia the AUST-02 shared strain is predominant in individuals attending CF centres in Queensland and Western Australia. M3L7 is a multidrug resistant sub-type of AUST-02 that was recently identified in a Queensland CF centre and was shown to be associated with poorer clinical outcomes. The main aim of this study was to resolve the relationship of the emergent M3L7 sub-type within the AUST-02 group of strains using whole genome sequencing., Results: A whole genome core phylogeny of 63 isolates indicated that M3L7 is a monophyletic sub-lineage within the context of the broader AUST-02 group. Relatively short branch lengths connected all of the M3L7 isolates. A phylogeny based on nucleotide polymorphisms present across the genome showed that the chronological estimation of the most recent common ancestor was around 2001 (± 3 years). SNP differences between sequential non-hypermutator M3L7 isolates collected 3-4 years apart from five patients suggested both continuous infection of the same strain and cross-infection of some M3L7 variants between patients. The majority of polymorphisms that were characteristic of M3L7 (i.e. acquired after divergence from all other AUST-02 isolates sequenced) were found to produce non-synonymous mutations in virulence and antibiotic resistance genes., Conclusions: M3L7 has recently diverged from a common ancestor, indicating descent from a single carrier at a CF treatment centre in Australia. Both adaptation to the lung and transmission of M3L7 between adults attending this centre may have contributed to its rapid dissemination. Further genomic investigations are required on multiple intra-sample isolates of this sub-type to decipher potential mechanisms which facilitates its epidemiological success.

Rationale: People with cystic fibrosis (CF) generate Pseudomonas aeruginosa in droplet nuclei during coughing. The use of surgical masks has been recommended in healthcare settings to minimize pathogen transmission between patients with CF., Objectives: To determine if face masks and cough etiquette reduce viable P. aeruginosa aerosolized during coughing., Methods: Twenty-five adults with CF and chronic P. aeruginosa infection were recruited. Participants performed six talking and coughing maneuvers, with or without face masks (surgical and N95) and hand covering the mouth when coughing (cough etiquette) in an aerosol-sampling device. An Andersen Cascade Impactor was used to sample the aerosol at 2 meters from each participant. Quantitative sputum and aerosol bacterial cultures were performed, and participants rated the mask comfort levels during the cough maneuvers., Measurements and Main Results: During uncovered coughing (reference maneuver), 19 of 25 (76%) participants produced aerosols containing P. aeruginosa, with a positive correlation found between sputum P. aeruginosa concentration (measured as cfu/ml) and aerosol P. aeruginosa colony-forming units. There was a reduction in aerosol P. aeruginosa load during coughing with a surgical mask, coughing with an N95 mask, and cough etiquette compared with uncovered coughing (P < 0.001). A similar reduction in total colony-forming units was observed for both masks during coughing; yet, participants rated the surgical masks as more comfortable (P = 0.013). Cough etiquette provided approximately half the reduction of viable aerosols of the mask interventions during voluntary coughing. Talking was a low viable aerosol-producing activity., Conclusions: Face masks reduce cough-generated P. aeruginosa aerosols, with the surgical mask providing enhanced comfort. Cough etiquette was less effective at reducing viable aerosols.

Background: Pulmonary exacerbations in cystic fibrosis (CF) remain poorly understood and treatment is usually targeted at Pseudomonas aeruginosa. Within Australia a predominant shared P. aeruginosa strain (AUST-02) is associated with greater treatment needs. This single centre study assessed temporal shared strain population dynamics during and after antibiotic treatment of exacerbations., Methods: Sputum was collected from 12 adult patients with a history of chronic AUST-02 infection at four time-points during and after treatment of an exacerbation. Forty-eight P. aeruginosa isolates within each sample underwent AUST-02 allele-specific PCR and SNP-based strain genotyping., Results: Various commonly shared Australian strains (AUST-01, 0.1%; AUST-02, 54.3%; AUST-06, 36.6%; AUST-07, 4.6%; AUST-11, 4.3%) and two unique strains (0.1%) were identified from 45 sputum samples (2160 isolates). Based on within-patient relative abundance of strains, a "single-strain infection" (n = 7) or "mixed-strain infection" (n = 5) was assigned to each patient. A significant temporal variation in the P. aeruginosa population composition was found for those with mixed-strain infection (P < 0.001). Patients with mixed-strain infections had more long-term treatment requirements than those with single-strain infection. Moreover, despite both groups having similar lung function at study entry, patients with single-strain infection had greater improvement in FEV 1 % predicted following their exacerbation treatment (P = 0.02)., Conclusion: Pulmonary exacerbations may reveal multiple, unrelated P. aeruginosa strains whose relative abundance with one another may change rapidly, in a sustained and unpredictable manner.

A Pseudomonas aeruginosa AUST-02 strain sub-type (M3L7) has been identified in Australia, infects the lungs of some people with cystic fibrosis and is associated with antibiotic resistance. Multiple clonal lineages may emerge during treatment with mutations in chromosomally encoded antibiotic resistance genes commonly observed. Here we describe the within-host diversity and antibiotic resistance of M3L7 during and after antibiotic treatment of an acute pulmonary exacerbation using whole genome sequencing and show both variation and shared mutations in important genes. Eleven isolates from an M3L7 population (n = 134) isolated over 3 months from an individual with cystic fibrosis underwent whole genome sequencing. A phylogeny based on core genome SNPs identified three distinct phylogenetic groups comprising two groups with higher rates of mutation (hypermutators) and one non-hypermutator group. Genomes were screened for acquired antibiotic resistance genes with the result suggesting that M3L7 resistance is principally driven by chromosomal mutations as no acquired mechanisms were detected. Small genetic variations, shared by all 11 isolates, were found in 49 genes associated with antibiotic resistance including frame-shift mutations (mexA, mexT), premature stop codons (oprD, mexB) and mutations in quinolone-resistance determining regions (gyrA, parE). However, whole genome sequencing also revealed mutations in 21 genes that were acquired following divergence of groups, which may also impact the activity of antibiotics and multi-drug efflux pumps. Comparison of mutations with minimum inhibitory concentrations of anti-pseudomonal antibiotics could not easily explain all resistance profiles observed. These data further demonstrate the complexity of chronic and antibiotic resistant P. aeruginosa infection where a multitude of co-existing genotypically diverse sub-lineages might co-exist during and after intravenous antibiotic treatment.

Background: Increased patient longevity and aggressive antibiotic treatment are thought to impact on the microbial composition of the airways of adults with cystic fibrosis (CF). In this study, we sought to determine if a temporal change in the airway microbiology of adults with CF has occurred over time., Methods: Longitudinal analysis of sputum microbiology results was undertaken on patients attending a large adult CF centre. Clinical status and health outcomes of transitioning patients were also assessed., Results: A decrease in the prevalence of Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia complex and Aspergillus spp. (p=0.001, p<0.001, p=0.002 and p<0.001, respectively) occurred. Improvements in lung function among transitioning patients infected with P. aeruginosa were observed., Conclusion: Overtime, a decline in the prevalence of many CF airway pathogens has occurred. Significantly, an incremental improvement in lung function was reported for transitioning patients with current P. aeruginosa infections., (Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Rationale: For patients with cystic fibrosis (CF), the use of inhaled antibiotics has become standard of care to suppress chronic Pseudomonas airways infection. There are limited antibiotic options formulated and approved for inhaled use and antibiotic efficacies attenuate over time, making additional inhaled antibiotic classes desirable. APT-1026 (levofloxacin inhalation solution, LIS) is a fluoroquinolone in development for management of chronic P. aeruginosa airways infection in patients with CF., Objectives: To compare the safety and efficacy of a 28-day course of treatment with LIS 240mg or placebo BID in persons ≥12years old with CF and chronic P. aeruginosa infection., Methods: A multinational, randomized (2:1), double-blinded study of LIS and placebo over 28days in CF patients ≥12years with chronic P. aeruginosa infection. Time to exacerbation was the primary endpoint. FEV1 (% predicted) and patient-reported quality of life were among secondary endpoints., Main Results: Baseline demographics for 330 subjects (LIS=220) were similar although significantly more patients randomized to LIS had experienced multiple exacerbations in the year prior to study entry. There was no statistically significant difference in protocol-defined pulmonary exacerbations between treatment arms. Relative change in FEV1% predicted from baseline was significantly greater for patients randomized to LIS compared to those randomized to placebo (mean difference 1.31%, p=0.01 [95% CI 0.27, 2.34%]). LIS was well-tolerated, with dysguesia the most frequent adverse event., Conclusions: LIS did not demonstrate a difference in time to next exacerbation when compared to placebo. Reasons for this result are discussed but may be due to an imbalance in the frequency of prior pulmonary exacerbations between the two groups. An improvement in FEV1 (% predicted) at 28days was observed and LIS was well tolerated. LIS is safe and has a potential role in the management of CF patients with chronic P. aeruginosa., (Copyright © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background and Objective: In cystic fibrosis (CF), chronic Pseudomonas aeruginosa infection is associated with increased morbidity, antibiotic treatments and mortality. By linking Australian CF registry data with a national microbiological data set, we examined the association between where treatment was delivered, its intensity and P. aeruginosa antibiotic resistance., Methods: Sputa were collected from paediatric and adult CF patients attending 18 Australian CF centres. P. aeruginosa antibiotic susceptibilities determined by local laboratories were correlated with clinical characteristics, treatment intensity and infection with strains commonly shared among Australian CF patients. Between-centre differences in treatment and antibiotic resistance were also compared., Results: Large variations in antibiotic usage, maintenance treatment practices and multi-antibiotic resistant P. aeruginosa (MARPA) prevalence exist between Australian CF centres, although the overall proportions of MARPA isolates were similar in paediatric and adult centres (31% vs 35%, P = 0.29). Among paediatric centres, MARPA correlated with intravenous antibiotic usage and the Australian state where treatment was delivered, while azithromycin, reduced lung function and treating state predicted intravenous antibiotic usage. In adult centres, body mass index (BMI) and treating state were associated with MARPA, while intravenous antibiotic use was predicted by gender, BMI, dornase-alpha, azithromycin, lung function and treating state. In adults, P. aeruginosa strains AUST-01 and AUST-02 independently predicted intravenous antibiotic usage., Conclusion: Increased treatment intensity in paediatric centres and the Australian state where treatment was received are both associated with greater risk of MARPA, but not worse clinical outcomes., (© 2015 Asian Pacific Society of Respirology.)

In cystic fibrosis (CF), Pseudomonas aeruginosa undergoes intra-strain genotypic and phenotypic diversification while establishing and maintaining chronic lung infections. As the clinical significance of these changes is uncertain, we investigated intra-strain diversity in commonly shared strains from CF patients to determine if specific gene mutations were associated with increased antibiotic resistance and worse clinical outcomes. Two-hundred-and-one P. aeruginosa isolates (163 represented a dominant Australian shared strain, AUST-02) from two Queensland CF centres over two distinct time-periods (2001-2002 and 2007-2009) underwent mexZ and lasR sequencing. Broth microdilution antibiotic susceptibility testing in a subset of isolates was also performed. We identified a novel AUST-02 subtype (M3L7) in adults attending a single Queensland CF centre. This M3L7 subtype was multi-drug resistant and had significantly higher antibiotic minimum inhibitory concentrations than other AUST-02 subtypes. Prospective molecular surveillance using polymerase chain reaction assays determined the prevalence of the 'M3L7' subtype at this centre during 2007-2009 (170 patients) and 2011 (173 patients). Three-year clinical outcomes of patients harbouring different strains and subtypes were compared. MexZ and LasR sequences from AUST-02 isolates were more likely in 2007-2009 than 2001-2002 to exhibit mutations (mexZ: odds ratio (OR) = 3.8; 95% confidence interval (CI): 1.1-13.5 and LasR: OR = 2.5; 95%CI: 1.3-5.0). Surveillance at the adult centre in 2007-2009 identified M3L7 in 28/509 (5.5%) P. aeruginosa isolates from 13/170 (7.6%) patients. A repeat survey in 2011 identified M3L7 in 21/519 (4.0%) P. aeruginosa isolates from 11/173 (6.4%) patients. The M3L7 subtype was associated with greater intravenous antibiotic and hospitalisation requirements, and a higher 3-year risk of death/lung transplantation, than other AUST-02 subtypes (adjusted hazard ratio [HR] = 9.4; 95%CI: 2.2-39.2) and non-AUST-02 strains (adjusted HR = 4.8; 95%CI: 1.4-16.2). This suggests ongoing microevolution of the shared CF strain, AUST-02, was associated with an emerging multi-drug resistant subtype and possibly poorer clinical outcomes.

Background: Person-to-person transmission is a potential pathway of Pseudomonas aeruginosa acquisition in cystic fibrosis. Reports of cross-infection of shared cystic-fibrosis-specific P. aeruginosa strains across large geographical distances are concerning. Therefore, we aimed to assess the extent to which patient movement between cystic fibrosis centres contributes to dissemination., Methods: We did a cross-sectional study to assess movement of patients with cystic fibrosis who were infected with P. aeruginosa between Sept 3, 2007, and June 16, 2010, at 18 Australian cystic fibrosis centres. We applied social network analysis to patient movement data from P. aeruginosa-infected patients to assess the role of patient mobility in P. aeruginosa genotype prevalence. We generated networks linking treatment centres based on the movement of patients attending adult and paediatric cystic fibrosis centres, and compared these with the movement of patients infected with all P. aeruginosa strains, unique strains, and predominant Australian shared strains (AUST-01 and AUST-02). We summarised connectivity using degree centrality, in-degree centrality, out-degree centrality, and k-core estimates. Infection control and surveillance practices were also assessed by use of a questionnaire., Findings: 983 patients (mean age 25 years [SD 10]; 551 [56%] male) provided 2887 P. aeruginosa isolates for ERIC-PCR genotyping, which yielded 531 distinct genotypes: 493 unique strains in 373 patients and 38 shared strains in 610 patients. AUST-01 infections were associated with higher in-degree centrality (p=0·004) and k-core (p=0·005) estimates and AUST-02 infections with higher degree centrality (p=0·002), out-degree centrality (p=0·002), and k-core (p=0·007) estimates for the previous health-care facilities; associations for the present cystic fibrosis centre were not significant. These findings were significant for adult patients (AUST-01 in-degree centrality p=0·004 and k-core p=0·005; AUST-02 degree centrality p=0·004, out-degree centrality p=0·003, and k-core p=0·007), but not for paediatric patients. By contrast, infections with unique strains were associated with a lower k-core estimate for the present cystic fibrosis centre overall (p<0·0001); this finding was significant in adults (p<0·0001), but not in paediatric patients., Interpretation: Our results show that the connectivity of cystic fibrosis centres, as measured by the movement of patients, seems to be an important risk factor for the acquisition of shared P. aeruginosa strain infections. These results show the importance of prioritising infection control interventions (eg, prospective molecular surveillance for shared P. aeruginosa strains, strict universal infection control precautions, and hospital design and ventilation) to limit P. aeruginosa cross-infection between patients with cystic fibrosis., Funding: Australian National Health and Medical Research Council; Children's Health Foundation Queensland; Office of Health and Medical Research, Queensland Health; European Respiratory Society-European Union; Australian Cystic Fibrosis Research Trust; Prince Charles Hospital Foundation; and Rotary Australia., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Inhaled antibiotics are standard of care for persons with cystic fibrosis (CF) and chronic Pseudomonas aeruginosa airway infection. APT-1026 (levofloxacin inhalation solution, LIS) is fluoroquinolone in development. We compared the safety and efficacy of LIS to tobramycin inhalation solution (TIS) in persons ≥12 years old with CF and chronic P. aeruginosa infection., Methods: This multinational, randomized (2:1), non-inferiority study compared LIS and TIS over three 28-day on/off cycles. Day 28 FEV(1) % predicted relative change was the primary endpoint. Time to exacerbation and patient-reported quality of life were among secondary endpoints., Results: Baseline demographics for 282 subjects were comparable. Non-inferiority was demonstrated (1.86% predicted mean FEV(1) difference [95% CI -0.66 to 4.39%]). LIS was well-tolerated, with dysgeusia (taste distortion) as the most frequent adverse event., Conclusions: LIS is a safe and effective therapy for the management of CF patients with chronic P. aeruginosa infection., (Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: We describe Pseudomonas aeruginosa acquisitions in children with cystic fibrosis (CF) aged ≤5-years, eradication treatment efficacy, and genotypic relationships between upper and lower airway isolates and strains from non-CF sources., Methods: Of 168 CF children aged ≤5-years in a bronchoalveolar lavage (BAL)-directed therapy trial, 155 had detailed microbiological results. Overall, 201/271 (74%) P. aeruginosa isolates from BAL and oropharyngeal cultures were available for genotyping, including those collected before and after eradication therapy., Results: Eighty-two (53%) subjects acquired P. aeruginosa, of which most were unique strains. Initial eradication success rate was 90%, but 36 (44%) reacquired P. aeruginosa, with genotypic substitutions more common in BAL (12/14) than oropharyngeal (3/11) cultures. Moreover, oropharyngeal cultures did not predict BAL genotypes reliably., Conclusions: CF children acquire environmental P. aeruginosa strains frequently. However, discordance between BAL and oropharyngeal strains raises questions over upper airway reservoirs and how to best determine eradication in non-expectorating children., (Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Carbapenemase phenotypic tests (inhibitor-based, modified Hodge and Carba NP) for Pseudomonas aeruginosa isolates from patients with cystic fibrosis (CF) were evaluated with PCR as a reference assay. Except for Carba NP, all methods demonstrated high false positives. Carba NP test presents a promising screening method for CF P. aeruginosa isolates., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to assess the prevalence of acquired carbapenemase genes amongst carbapenem non-susceptible Pseudomonas aeruginosa isolates in Australian patients with cystic fibrosis (CF). Cross-sectional molecular surveillance for acquired carbapenemase genes was performed on CF P. aeruginosa isolates from two isolate banks comprising: (i) 662 carbapenem resistant P. aeruginosa isolates from 227 patients attending 10 geographically diverse Australian CF centres (2007-2009), and (ii) 519 P. aeruginosa isolates from a cohort of 173 adult patients attending one Queensland CF clinic in 2011. All 1189 P. aeruginosa isolates were tested by polymerase chain reaction (PCR) protocols targeting ten common carbapenemase genes, as well the Class 1 integron intI1 gene and the aadB aminoglycoside resistance gene. No carbapenemase genes were identified among all isolates tested. The intI1 and aadB genes were frequently detected and were significantly associated with the AUST-02 strain (OR 24.6, 95% CI 9.3-65.6; p < 0.0001) predominantly from Queensland patients. Despite the high prevalence of carbapenem resistance in P. aeruginosa in Australian patients with CF, no acquired carbapenemase genes were detected in the study, suggesting chromosomal mutations remain the key resistance mechanism in CF isolates. Systematic surveillance for carbapenemase-producing P. aeruginosa in CF by molecular surveillance is ongoing.

Background: Primary defects in host immune responses have been hypothesised to contribute towards an inability of subjects with cystic fibrosis (CF) to effectively clear pulmonary infections. Innate T-lymphocytes provide rapid pathogen-specific responses prior to the development of classical MHC class I and II restricted T-cell responses and are essential to the initial control of pulmonary infection. We aimed to examine the relationship between peripheral blood lymphocyte phenotype and clinical outcomes in adults with CF., Methods: We studied 41 subjects with CF and 22, age matched, non-smoking healthy control subjects. Lymphocytes were extracted from peripheral blood samples and phenotyped by flow-cytometry. Lymphocyte phenotype was correlated with sputum microbiology and clinical parameters., Results: In comparison to healthy control subjects, mucosal associated invariant T (MAIT)-lymphocytes were significantly reduced in the peripheral blood of subjects with CF (1.1% versus 2.0% of T-lymphocytes, P = 0.002). MAIT cell concentration was lowest in CF subjects infected with P. aeruginosa and in subjects receiving treatment for a pulmonary exacerbation. Furthermore a reduced MAIT cell concentration correlated with severity of lung disease., Conclusion: Reduced numbers of MAIT cells in subjects with CF were associated with P. aeruginosa pulmonary infection, pulmonary exacerbations and more severe lung disease. These findings provide the impetus for future studies examining the utility of MAIT cells in immunotherapies and vaccine development. Longitudinal studies of MAIT cells as biomarkers of CF pulmonary infection are awaited.

Background: Person-to-person transmission of respiratory pathogens, including Pseudomonas aeruginosa, is a challenge facing many cystic fibrosis (CF) centres. Viable P aeruginosa are contained in aerosols produced during coughing, raising the possibility of airborne transmission., Methods: Using purpose-built equipment, we measured viable P aeruginosa in cough aerosols at 1, 2 and 4 m from the subject (distance) and after allowing aerosols to age for 5, 15 and 45 min in a slowly rotating drum to minimise gravitational settling and inertial impaction (duration). Aerosol particles were captured and sized employing an Anderson Impactor and cultured using conventional microbiology. Sputum was also cultured and lung function and respiratory muscle strength measured., Results: Nineteen patients with CF, mean age 25.8 (SD 9.2) years, chronically infected with P aeruginosa, and 10 healthy controls, 26.5 (8.7) years, participated. Viable P aeruginosa were detected in cough aerosols from all patients with CF, but not from controls; travelling 4 m in 17/18 (94%) and persisting for 45 min in 14/18 (78%) of the CF group. Marked inter-subject heterogeneity of P aeruginosa aerosol colony counts was seen and correlated strongly (r=0.73-0.90) with sputum bacterial loads. Modelling decay of viable P aeruginosa in a clinic room suggested that at the recommended ventilation rate of two air changes per hour almost 50 min were required for 90% to be removed after an infected patient left the room., Conclusions: Viable P aeruginosa in cough aerosols travel further and last longer than recognised previously, providing additional evidence of airborne transmission between patients with CF., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Molecular typing is integral for identifying Pseudomonas aeruginosa strains that may be shared between patients with cystic fibrosis (CF). We conducted a side-by-side comparison of two P. aeruginosa genotyping methods utilising informative-single nucleotide polymorphism (SNP) methods; one targeting 10 P. aeruginosa SNPs and using real-time polymerase chain reaction technology (HRM10SNP) and the other targeting 20 SNPs and based on the Sequenom MassARRAY platform (iPLEX20SNP)., Methods: An in-silico analysis of the 20 SNPs used for the iPLEX20SNP method was initially conducted using sequence type (ST) data on the P. aeruginosa PubMLST website. A total of 506 clinical isolates collected from patients attending 11 CF centres throughout Australia were then tested by both the HRM10SNP and iPLEX20SNP assays. Type-ability and discriminatory power of the methods, as well as their ability to identify commonly shared P. aeruginosa strains, were compared., Results: The in-silico analyses showed that the 1401 STs available on the PubMLST website could be divided into 927 different 20-SNP profiles (D-value = 0.999), and that most STs of national or international importance in CF could be distinguished either individually or as belonging to closely related single- or double-locus variant groups. When applied to the 506 clinical isolates, the iPLEX20SNP provided better discrimination over the HRM10SNP method with 147 different 20-SNP and 92 different 10-SNP profiles observed, respectively. For detecting the three most commonly shared Australian P. aeruginosa strains AUST-01, AUST-02 and AUST-06, the two methods were in agreement for 80/81 (98.8%), 48/49 (97.8%) and 11/12 (91.7%) isolates, respectively., Conclusions: The iPLEX20SNP is a superior new method for broader SNP-based MLST-style investigations of P. aeruginosa. However, because of convenience and availability, the HRM10SNP method remains better suited for clinical microbiology laboratories that only utilise real-time PCR technology and where the main interest is detection of the most highly-prevalent P. aeruginosa CF strains within Australian clinics.

Recent molecular-typing studies suggest cross-infection as one of the potential acquisition pathways for Pseudomonas aeruginosa in patients with cystic fibrosis (CF). In Australia, there is only limited evidence of unrelated patients sharing indistinguishable P. aeruginosa strains. We therefore examined the point-prevalence, distribution, diversity and clinical impact of P. aeruginosa strains in Australian CF patients nationally. 983 patients attending 18 Australian CF centres provided 2887 sputum P. aeruginosa isolates for genotyping by enterobacterial repetitive intergenic consensus-PCR assays with confirmation by multilocus sequence typing. Demographic and clinical details were recorded for each participant. Overall, 610 (62%) patients harboured at least one of 38 shared genotypes. Most shared strains were in small patient clusters from a limited number of centres. However, the two predominant genotypes, AUST-01 and AUST-02, were widely dispersed, being detected in 220 (22%) and 173 (18%) patients attending 17 and 16 centres, respectively. AUST-01 was associated with significantly greater treatment requirements than unique P. aeruginosa strains. Multiple clusters of shared P. aeruginosa strains are common in Australian CF centres. At least one of the predominant and widespread genotypes is associated with increased healthcare utilisation. Longitudinal studies are now needed to determine the infection control implications of these findings.

Shared strains of Pseudomonas aeruginosa are now well recognized in people with cystic fibrosis (CF), and suitable P. aeruginosa laboratory typing tools are pivotal to understanding their clinical significance and guiding infection control policies in CF clinics. We therefore compared a single-nucleotide polymorphism (SNP)-based typing method using Sequenom iPLEX matrix-assisted laser desorption ionization with time-of-flight mass spectrometry (MALDI-TOF MS) with typing methods used routinely by our laboratory. We analysed 617 P. aeruginosa isolates that included 561 isolates from CF patients collected between 2001 and 2009 in two Brisbane CF clinics and typed previously by enterobacterial repetitive intergenic consensus (ERIC)-PCR, as well as 56 isolates from non-CF patients analysed previously by multilocus sequence typing (MLST). The isolates were tested using a P. aeruginosa Sequenom iPLEX MALDI-TOF (PA iPLEX) method comprising two multiplex reactions, a 13-plex and an 8-plex, to characterize 20 SNPs from the P. aeruginosa housekeeping genes acsA, aroE, guaA, mutL, nuoD, ppsA and trpE. These 20 SNPs were employed previously in a real-time format involving 20 separate assays in our laboratory. The SNP analysis revealed 121 different SNP profiles for the 561 CF isolates. Overall, there was at least 96% agreement between the ERIC-PCR and SNP analyses for all predominant shared strains among patients attending our CF clinics: AUST-01, AUST-02 and AUST-06. For the less frequently encountered shared strain AUST-07, 6/25 (24%) ERIC-PCR profiles were misidentified initially as AUST-02 or as unique, illustrating the difficulty of gel-based analyses. SNP results for the 56 non-CF isolates were consistent with previous MLST data. Thus, the PA iPLEX format provides an attractive high-throughput alternative to ERIC-PCR for large-scale investigations of shared P. aeruginosa strains.

Rationale: Risk of infection with Pseudomonas aeruginosa in cystic fibrosis (CF) may be associated with environmental factors., Objectives: To determine whether residential location is associated with risk of first acquisition of P. aeruginosa., Methods: We performed bronchoalveolar lavage and upper airway cultures in children newly diagnosed with CF to identify infection with P. aeruginosa during infancy and early childhood. Children were assessed according to their residence in a regional or metropolitan area. Multilocus sequence typing was used to determine P. aeruginosa genotype. An environmental questionnaire was also administered., Measurements and Main Results: A total of 105 of 120 (87.5%) infants diagnosed with CF were included in this study. Diagnosis in 65 infants (61.9%) followed newborn screening at mean age of 4.6 weeks. Sixty subjects (57.1%) were homozygous ΔF508, and 47 (44.8%) were female. Fifty-five (52.3%) infants were regional, of whom 26 (47.3%), compared with 9 of 50 (18.0%) metropolitan children, acquired infection with P. aeruginosa (odds ratio, 4.084; 95% confidence interval, 1.55-11.30). Age at acquisition was similar (regional: median, 2.31 yr; range, 0.27-5.96 yr; metropolitan: median, 3.10 yr, range, 0.89-3.70 yr). Strain typing identified P. aeruginosa genotypes often encountered in different ecological settings and little evidence of cross-infection. Ninety questionnaires (85.7%) were completed. Those who acquired P. aeruginosa were more likely to be living in a household that used water sprinkler systems (P = 0.032), but no differences were identified to explain increased risk of acquisition of P. aeruginosa in regional children., Conclusions: Geographical difference in residence of children with CF was associated with increased risk of first acquisition of P. aeruginosa, usually with strains associated with the environment rather than with cross-infection.

Pseudomonas aeruginosa is an opportunistic pathogen and an important cause of infection, particularly amongst cystic fibrosis (CF) patients. While specific strains capable of patient-to-patient transmission are known, many infections appear to be caused by unique and unrelated strains. There is a need to understand the relationship between strains capable of colonising the CF lung and the broader set of P. aeruginosa isolates found in natural environments. Here we report the results of a multilocus sequence typing (MLST)-based study designed to understand the genetic diversity and population structure of an extensive regional sample of P. aeruginosa isolates from South East Queensland, Australia. The analysis is based on 501 P. aeruginosa isolates obtained from environmental, animal and human (CF and non-CF) sources with particular emphasis on isolates from the Lower Brisbane River and isolates from CF patients obtained from the same geographical region. Overall, MLST identified 274 different sequence types, of which 53 were shared between one or more ecological settings. Our analysis revealed a limited association between genotype and environment and evidence of frequent recombination. We also found that genetic diversity of P. aeruginosa in Queensland, Australia was indistinguishable from that of the global P. aeruginosa population. Several CF strains were encountered frequently in multiple ecological settings; however, the most frequently encountered CF strains were confined to CF patients. Overall, our data confirm a non-clonal epidemic structure and indicate that most CF strains are a random sample of the broader P. aeruginosa population. The increased abundance of some CF strains in different geographical regions is a likely product of chance colonisation events followed by adaptation to the CF lung and horizontal transmission among patients.

Pseudomonas aeruginosa genotyping relies mainly upon DNA fingerprinting methods, which can be subjective, expensive and time-consuming. The detection of at least three different clonal P. aeruginosa strains in patients attending two cystic fibrosis (CF) centres in a single Australian city prompted the design of a non-gel-based PCR method to enable clinical microbiology laboratories to readily identify these clonal strains. We designed a detection method utilizing heat-denatured P. aeruginosa isolates and a ten-single-nucleotide polymorphism (SNP) profile. Strain differences were detected by SYBR Green-based real-time PCR and high-resolution melting curve analysis (HRM10SNP assay). Overall, 106 P. aeruginosa sputum isolates collected from 74 patients with CF, as well as five reference strains, were analysed with the HRM10SNP assay, and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE). The HRM10SNP assay accurately identified all 45 isolates as members of one of the three major clonal strains characterized by PFGE in two Brisbane CF centres (Australian epidemic strain-1, Australian epidemic strain-2 and P42) from 61 other P. aeruginosa strains from Australian CF patients and two representative overseas epidemic strain isolates. The HRM10SNP method is simple, is relatively inexpensive and can be completed in <3 h. In our setting, it could be made easily available for clinical microbiology laboratories to screen for local P. aeruginosa strains and to guide infection control policies. Further studies are needed to determine whether the HRM10SNP assay can also be modified to detect additional clonal strains that are prevalent in other CF centres., (© 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.)

Pseudomonas aeruginosa is associated with infectious endometritis in horses. Although infectious endometritis is often considered a venereal infection, there is relatively limited genotypic-based evidence to support this mode of transmission. The study sought to determine the relatedness between genital P. aeruginosa isolates collected from a limited geographical region using molecular strain typing. Enterobacterial repetitive intergenic consensus PCR typing was performed on 93 isolates collected between 2005 and 2009 from 2058 thoroughbred horses (including 18 stallions) at 66 studs. While P. aeruginosa was not detected in the stallions, 53/93 (57%) mares harbouring P. aeruginosa had clonally related strains, which included a single dominant genotype detected in 42 (45%) mares from 13 different studs. These novel findings suggest that most equine genital P. aeruginosa infections in this region may have been acquired from mechanisms other than direct horse to horse transmission. Instead, other potential acquisition pathways, as well as strain specific adaptation to the equine genital tract, should be investigated., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Monitoring the emergence and transmission of Pseudomonas aeruginosa strains among cystic fibrosis (CF) patients is important for infection control in CF centers internationally. A recently developed multilocus sequence typing (MLST) scheme is used for epidemiologic analyses of P. aeruginosa outbreaks; however, little is known about its suitability for isolates from CF patients compared with that of pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). As part of a prevalence study of P. aeruginosa strains in Australian CF clinics, we compared the discriminatory power and concordance of ERIC-PCR, PFGE, and MLST among 93 CF sputum and 11 control P. aeruginosa isolates. PFGE and MLST analyses were also performed on 30 paired isolates collected 85 to 354 days apart from 30 patients attending two CF centers separated by 3,600 kilometers in order to detect within-host evolution. Each of the three methods displayed high levels of concordance and discrimination; however, overall lower discrimination was seen with ERIC-PCR than with MLST and PFGE. Analysis of the 50 ERIC-PCR types yielded 54 PFGE types, which were related by ≤ 6 band differences, and 59 sequence types, which were classified into 7 BURST groups and 42 singletons. MLST also proved useful for detecting novel and known strains and for inferring relatedness among unique PFGE types. However, 47% of the paired isolates produced PFGE patterns that within 1 year differed by one to five bands, whereas with MLST all paired isolates remained identical. MLST thus represents a categorical analysis tool with resolving power similar to that of PFGE for typing P. aeruginosa. Its focus on highly conserved housekeeping genes is particularly suited for long-term clinical monitoring and detecting novel strains.

Pseudomonas aeruginosa is an important cause of pulmonary infection in cystic fibrosis (CF). Its correct identification ensures effective patient management and infection control strategies. However, little is known about how often CF sputum isolates are falsely identified as P. aeruginosa. We used P. aeruginosa-specific duplex real-time PCR assays to determine if 2,267 P. aeruginosa sputum isolates from 561 CF patients were correctly identified by 17 Australian clinical microbiology laboratories. Misidentified isolates underwent further phenotypic tests, amplified rRNA gene restriction analysis, and partial 16S rRNA gene sequence analysis. Participating laboratories were surveyed on how they identified P. aeruginosa from CF sputum. Overall, 2,214 (97.7%) isolates from 531 (94.7%) CF patients were correctly identified as P. aeruginosa. Further testing with the API 20NE kit correctly identified only 34 (59%) of the misidentified isolates. Twelve (40%) patients had previously grown the misidentified species in their sputum. Achromobacter xylosoxidans (n = 21), Stenotrophomonas maltophilia (n = 15), and Inquilinus limosus (n = 4) were the species most commonly misidentified as P. aeruginosa. Overall, there were very low rates of P. aeruginosa misidentification among isolates from a broad cross section of Australian CF patients. Additional improvements are possible by undertaking a culture history review, noting colonial morphology, and performing stringent oxidase, DNase, and colistin susceptibility testing for all presumptive P. aeruginosa isolates. Isolates exhibiting atypical phenotypic features should be evaluated further by additional phenotypic or genotypic identification techniques.

Phenotypic identification of Gram-negative bacteria from respiratory specimens of patients with cystic fibrosis carries a high risk of misidentification. Molecular identification techniques that use single-gene targets are also susceptible to error, including cross-reaction issues with other Gram-negative organisms. In this study, we have designed a Pseudomonas aeruginosa duplex real-time polymerase chain reaction (PCR) (PAduplex) assay targeting the ecfX and the gyrB genes. The PAduplex was evaluated against a panel of 91 clinical and environmental isolates that were presumptively identified as P. aeruginosa. The results were compared with those obtained using a commercial biochemical identification kit and several other P. aeruginosa PCR assays. The results showed that the PAduplex assay is highly suitable for routine identification of P. aeruginosa isolates from clinical or environmental samples. The 2-target format provides simultaneous confirmation of P. aeruginosa identity where both the ecfX and gyrB PCR reactions are positive and may also reduce the potential for false negatives caused by sequence variation in primer or probe targets.

Aim: Concentrations of antimicrobials below minimum inhibitory concentration (subMIC) may reduce the production by Pseudomonas aeruginosa of virulence factors such as elastase. We sought to determine whether the reduction in elastase production may be mediated by a reduction in acyl-homoserine lactones., Methods: Pseudomonas aeruginosa in broth was exposed to three conditions for ceftazidime and tobramycin: control, 6% MIC and 25% MIC. Elastase was assayed using elastin congo red. N-(3-Oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl-homoserine lactone (C4-HSL) were assayed using biosensor Escherichia coli., Results: Elastase was unchanged with ceftazidime. Elastase was reduced by 16% at 6% MIC tobramycin and reduced by 70% at 25% MIC tobramycin (P<0.0001). As a percentage of control, C12-HSL was mean 69.4% (SEM 7.3%) at 6% MIC tobramycin, and 31.7% (3.3%) at 25% MIC tobramycin (P=0.0001). C12-HSL was 78.9% (5.3%) at 6% MIC ceftazidime and was 29.7% (1.8%) at 25% MIC ceftazidime (P=0.0001). Both ceftazidime and tobramycin were associated with reduced C4-HSL at 6% MIC and 25% MIC (P<0.03)., Conclusions: SubMIC tobramycin but not ceftazidime reduced elastase production by P. aeruginosa. In contrast, subMIC concentrations of both antimicrobials reduced C12-HSL and C4-HSL. It is unlikely that reduced HSL is the sole explanation for the reduction in elastase.

In this study, the suitability of two repetitive-element-based PCR (rep-PCR) assays, enterobacterial repetitive intergenic consensus (ERIC)-PCR and BOX-PCR, to rapidly characterize Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis (CF) was examined. ERIC-PCR utilizes paired sequence-specific primers and BOX-PCR a single primer that target highly conserved repetitive elements in the P. aeruginosa genome. Using these rep-PCR assays, 163 P. aeruginosa isolates cultured from sputa collected from 50 patients attending an adult CF clinic and 50 children attending a paediatric CF clinic were typed. The results of the rep-PCR assays were compared to the results of PFGE. All three assays revealed the presence of six major clonal groups shared by multiple patients attending either of the CF clinics, with the dominant clonal group infecting 38 % of all patients. This dominant clonal group was not related to the dominant clonal group detected in Sydney or Melbourne (pulsotype 1), nor was it related to the dominant groups detected in the UK. In all, PFGE and rep-PCR identified 58 distinct clonal groups, with only three of these shared between the two clinics. The results of this study showed that both ERIC-PCR and BOX-PCR are rapid, highly discriminatory and reproducible assays that proved to be powerful surveillance screening tools for the typing of clinical P. aeruginosa isolates recovered from patients with CF.

Despite recent reports of clonal strains of Pseudomonas aeruginosa in cystic fibrosis (CF) units, the need for routine microbiological surveillance remains contentious. Sputum was collected prospectively from productive patients attending the regional paediatric and adult CF units in Brisbane, Australia. All P. aeruginosa isolates were typed using pulsed-field gel electrophoresis. Spirometry, anthropometrics, hospitalisations and antibiotic sensitivity data were recorded. The first 100 sputum samples (first 50 patients at each clinic) harboured 163 isolates of P. aeruginosa. A total of 39 patients shared a common strain (pulsotype 2), 20 patients shared a strain with at least one other patient and 41 patients harboured unique strains. Eight patients shared a strain identical to a previously reported Australian transmissible strain (pulsotype 1). Compared with the unique strain group, patients harbouring pulsotype 2 were younger and had poorer lung function. Treatment requirements were similar in these two groups, as were the rates of multiresistance. In conclusion, 59% of patients harboured a clonal strain, supporting the need for routine microbiological surveillance. In contrast to previously described clonal strains, the dominant pulsotype was indistinguishable from nonclonal strains with respect to both colonial morphology and multiresistance. The clinical significance of clonal strains remains uncertain and requires longitudinal study.

Background: We hypothesized that increased resting energy expenditure in adults with cystic fibrosis was related to chronic inflammation secondary to pulmonary infection and could be modified by treatment of the underlying infection., Method: To determine the relationship between resting energy expenditure and the inflammatory and metabolic responses, we studied 22 adults with cystic fibrosis and chronic Pseudomonas aeruginosa infection before and after treatment of a respiratory exacerbation. Resting energy expenditure was measured by indirect calorimetry. Spirometry and circulating concentrations of C-reactive protein, neutrophil elastase alpha1-antiproteinase complex, catecholamines, non-esterified fatty acids and glycerol were determined., Results: The mean (95% confidence interval)% predicted FEV1 was 28.5% (20.6, 36.4) and mean body weight 50.7 kg (47.4, 54.1). Following treatment, 1-s forced expiratory volume (FEV1) and weight increased, while C-reactive protein (P<0.0001) and neutrophil elastase alpha1-antiproteinase complex concentrations (P<0.0001) were reduced. Resting energy expenditure decreased from 6.8 (6.3, 7.2) to 6.25 (5.9, 6.6) MJ day-1 by day 15 (P<0.001). Changes in resting energy expenditure and C-reactive protein were related (r = 0.66, P< 0.0001). Weight gain was inversely related to resting energy expenditure (r = 0.43, P = 0.02) and unrelated to energy intake (r = 0.02, P = 0.47). Post-treatment reduction in norepinephrine was related to changes in heart rate (r = 0.57, P<0.01), resting energy expenditure (r = 0.51, P = 0.001) and non-esterified fatty acids (r = 0.42, P< 0.05)., Conclusions: A parallel reduction in the host inflammatory and catabolic responses followed treatment of a respiratory exacerbation and may have contributed to weight gain.