Pseudomonas aeruginosa adaptation and diversification in the non-cystic fibrosis bronchiectasis lung.

To characterise Pseudomonas aeruginosa populations during chronic lung infections of non-cystic fibrosis bronchiectasis patients, we used whole-genome sequencing to 1) assess the diversity of P. aeruginosa and the prevalence of multilineage infections; 2) seek evidence for cross-infection or common source acquisition; and 3) characterise P. aeruginosa adaptations.189 isolates, obtained from the sputa of 91 patients attending 16 adult bronchiectasis centres in the UK, were whole-genome sequenced.Bronchiectasis isolates were representative of the wider P. aeruginosa population. Of 24 patients from whom multiple isolates were examined, there were seven examples of multilineage infections, probably arising from multiple infection events. The number of nucleotide variants between genomes of isolates from different patients was in some cases similar to the variations observed between isolates from individual patients, implying the possible occurrence of cross-infection or common source acquisition.Our data indicate that during infections of bronchiectasis patients, P. aeruginosa populations adapt by accumulating loss-of-function mutations, leading to changes in phenotypes including different modes of iron acquisition and variations in biofilm-associated polysaccharides. The within-population diversification suggests that larger scale longitudinal surveillance studies will be required to capture cross-infection or common source acquisition events at an early stage.

Inhaled colistin in patients with bronchiectasis and chronic Pseudomonas aeruginosa infection.

RATIONALE: Chronic infection with Pseudomonas aeruginosa is associated with an increased exacerbation frequency, a more rapid decline in lung function, and increased mortality in patients with bronchiectasis. OBJECTIVES: To perform a randomized placebo-controlled study assessing the efficacy and safety of inhaled colistin in patients with bronchiectasis and chronic P. aeruginosa infection. METHODS: Patients with bronchiectasis and chronic P. aeruginosa infection were enrolled within 21 days of completing a course of antipseudomonal antibiotics for an exacerbation. Participants were randomized to receive colistin (1 million IU; n = 73) or placebo (0.45% saline; n = 71) via the I-neb twice a day, for up to 6 months. MEASUREMENTS AND MAIN RESULTS: The primary endpoint was time to exacerbation. Secondary endpoints included time to exacerbation based on adherence recorded by the I-neb, P. aeruginosa bacterial density, quality of life, and safety parameters. All analyses were on the intention-to-treat population. Median time (25% quartile) to exacerbation was 165 (42) versus 111 (52) days in the colistin and placebo groups, respectively (P = 0.11). In adherent patients (adherence quartiles 2-4), the median time to exacerbation was 168 (65) versus 103 (37) days in the colistin and placebo groups, respectively (P = 0.038). P. aeruginosa density was reduced after 4 (P = 0.001) and 12 weeks (P = 0.008) and the St. George's Respiratory Questionnaire total score was improved after 26 weeks (P = 0.006) in the colistin versus placebo patients, respectively. There were no safety concerns. CONCLUSIONS: Although the primary endpoint was not reached, this study shows that inhaled colistin is a safe and effective treatment in adherent patients with bronchiectasis and chronic P. aeruginosa infection. Clinical trial registered with http://www.isrctn.org/ (ISRCTN49790596).

Whole-genome sequencing to identify transmission of Mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study.

BACKGROUND: Increasing numbers of individuals with cystic fibrosis are becoming infected with the multidrug-resistant non-tuberculous mycobacterium (NTM) Mycobacterium abscessus, which causes progressive lung damage and is extremely challenging to treat. How this organism is acquired is not currently known, but there is growing concern that person-to-person transmission could occur. We aimed to define the mechanisms of acquisition of M abscessus in individuals with cystic fibrosis. METHOD: Whole genome sequencing and antimicrobial susceptibility testing were done on 168 consecutive isolates of M abscessus from 31 patients attending an adult cystic fibrosis centre in the UK between 2007 and 2011. In parallel, we undertook detailed environmental testing for NTM and defined potential opportunities for transmission between patients both in and out of hospital using epidemiological data and social network analysis. FINDINGS: Phylogenetic analysis revealed two clustered outbreaks of near-identical isolates of the M abscessus subspecies massiliense (from 11 patients), differing by less than ten base pairs. This variation represents less diversity than that seen within isolates from a single individual, strongly indicating between-patient transmission. All patients within these clusters had numerous opportunities for within-hospital transmission from other individuals, while comprehensive environmental sampling, initiated during the outbreak, failed to detect any potential point source of NTM infection. The clusters of M abscessus subspecies massiliense showed evidence of transmission of mutations acquired during infection of an individual to other patients. Thus, isolates with constitutive resistance to amikacin and clarithromycin were isolated from several individuals never previously exposed to long-term macrolides or aminoglycosides, further indicating cross-infection. INTERPRETATION: Whole genome sequencing has revealed frequent transmission of multidrug resistant NTM between patients with cystic fibrosis despite conventional cross-infection measures. Although the exact transmission route is yet to be established, our epidemiological analysis suggests that it could be indirect. FUNDING: The Wellcome Trust, Papworth Hospital, NIHR Cambridge Biomedical Research Centre, UK Health Protection Agency, Medical Research Council, and the UKCRC Translational Infection Research Initiative.

Genomic variation among contemporary Pseudomonas aeruginosa isolates from chronically infected cystic fibrosis patients.

The airways of individuals with cystic fibrosis (CF) often become chronically infected with unique strains of the opportunistic pathogen Pseudomonas aeruginosa. Several lines of evidence suggest that the infecting P. aeruginosa lineage diversifies in the CF lung niche, yet so far this contemporary diversity has not been investigated at a genomic level. In this work, we sequenced the genomes of pairs of randomly selected contemporary isolates sampled from the expectorated sputum of three chronically infected adult CF patients. Each patient was infected by a distinct strain of P. aeruginosa. Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified in the DNA common to the paired isolates from different patients. The paired isolates from one patient differed due to just 1 SNP and 8 indels. The paired isolates from a second patient differed due to 54 SNPs and 38 indels. The pair of isolates from the third patient both contained a mutS mutation, which conferred a hypermutator phenotype; these isolates cumulatively differed due to 344 SNPs and 93 indels. In two of the pairs of isolates, a different accessory genome composition, specifically integrated prophage, was identified in one but not the other isolate of each pair. We conclude that contemporary isolates from a single sputum sample can differ at the SNP, indel, and accessory genome levels and that the cross-sectional genomic variation among coeval pairs of P. aeruginosa CF isolates can be comparable to the variation previously reported to differentiate between paired longitudinally sampled isolates.

Guidelines for the diagnosis and antibiotic treatment of endocarditis in adults: a report of the Working Party of the British Society for Antimicrobial Chemotherapy.

The BSAC guidelines on treatment of infectious endocarditis (IE) were last published in 2004. The guidelines presented here have been updated and extended to reflect developments in diagnostics, new trial data and the availability of new antibiotics. The aim of these guidelines, which cover both native valve and prosthetic valve endocarditis, is to standardize the initial investigation and treatment of IE. An extensive review of the literature using a number of different search criteria has been carried out and cited publications used to support any changes we have made to the existing guidelines. Publications referring to in vitro or animal models have only been cited if appropriate clinical data are not available. Randomized, controlled trials suitable for the development of evidenced-based guidelines in this area are still lacking and therefore a consensus approach has again been adopted for most recommendations; however, we have attempted to grade the evidence, where possible. The guidelines have also been extended by the inclusion of sections on clinical diagnosis, echocardiography and surgery.

The Pseudomonas aeruginosa generalized transducing phage phiPA3 is a new member of the phiKZ-like group of 'jumbo' phages, and infects model laboratory strains and clinical isolates from cystic fibrosis patients.

Pseudomonas aeruginosa is an important pathogen in cystic fibrosis patients, and a model organism for the study of nosocomially acquired infections, biofilms and intrinsic multidrug resistance. In this study we characterize ϕPA3, a new generalized transducing bacteriophage for P. aeruginosa. ϕPA3 transduced chromosomal mutations between PAO1 strains, and infected multiple P. aeruginosa clinical isolates as well as the P. aeruginosa model laboratory strains PAK and PA14. Electron microscopy imaging was used to classify ϕPA3 in the order Caudovirales and the family Myoviridae. The genome of ϕPA3 was sequenced and found to contain 309,208 bp, the second-largest bacteriophage currently deposited in GenBank. The genome contains 378 ORFs and five tRNAs. Many ORF products in the ϕPA3 genome are similar to proteins encoded by P. aeruginosa phage ϕKZ and Pseudomonas chlororaphis phage 201ϕ2-1, and so ϕPA3 was classified genetically as a member of the ϕKZ-like group of phages. This is the first report of a member of this group of phages acting as a generalized transducer. Given its wide host range, high transduction efficiency and large genome size, the 'jumbo' phage ϕPA3 could be a powerful tool in functional genomic analysis of diverse P. aeruginosa strains of fundamental and clinical importance.

Impact of Pseudomonas aeruginosa genomic instability on the application of typing methods for chronic cystic fibrosis infections.

The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is widespread among cystic fibrosis (CF) patients in the United Kingdom and has emerged recently in North America. In this study, we report the analysis of 24 "anomalous" CF isolates of P. aeruginosa that produced inconsistent results with regard to either pulsed-field gel electrophoresis (PFGE) or PCR tests for the LES. We used a new typing method, the ArrayTube genotyping system, to determine that of the 24 anomalous isolates tested, 13 were confirmed as the LES. LES isolates could not be clearly distinguished from non-LES isolates by two other commonly used genetic fingerprinting tests, randomly amplified polymorphic DNA (RAPD) analysis and BOX-PCR, and varied considerably in their carriage of LES genomic islands and prophages. The genomic instability of the LES suggests that identification of this emerging transmissible strain could be a challenging task, and it questions whether discrimination is always a desirable feature of bacterial typing methods in the context of chronic CF infections.

Comparison of methods to test antibiotic combinations against heterogeneous populations of multiresistant Pseudomonas aeruginosa from patients with acute infective exacerbations in cystic fibrosis.

Multiresistant Pseudomonas aeruginosa isolates can chronically infect patients with cystic fibrosis. Acute infective exacerbations are treated with combinations of two antipseudomonal antibiotics. Patients may respond clinically even if the bacteria are resistant, possibly due to antimicrobial synergy. The challenge for testing for synergy in vitro is that there is no standardized method, and the antibiotic susceptibility in a population of P. aeruginosa isolates in a single sputum sample can vary. We therefore compared (i) antibiotic combinations with different examples of resistant bacteria from the same sputum sample and (ii) the results of synergy testing by different methods. Antibiotic synergy was tested by using resistant P. aeruginosa isolates recovered from sputum samples taken just before the start of treatment for an acute infective exacerbation. Several examples of each morphotype of P. aeruginosa were tested by cidal checkerboard, time-kill curve, and multiple-combination bactericidal testing. The isolates were typed by pulsed-field gel electrophoresis (PFGE). The results were compared with the clinical and microbiological responses to 14 days of antibiotic treatment. Forty-four resistant isolates from nine patients were tested. Some P. aeruginosa isolates with the same morphotype and PFGE pulsotype had different results by synergy testing. There was a poor correlation between the results of the different methods of synergy testing, and no one method would have predicted the response to treatment in all patients. The in vitro effects of antibiotic combinations against different isolates from the same sputum sample can vary, and the results depend on the methodology used. The role of combination testing for the treatment of antibiotic-resistant P. aeruginosa in acute exacerbations of chronic infection in patients with cystic fibrosis needs to be reviewed.

Recent advances in the microbiology of respiratory tract infection in cystic fibrosis.

INTRODUCTION: Infection is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). Research on CF infection has highlighted differences from other respiratory infections--both in the range and the nature of the organisms--especially in chronic infection. This is a rapidly advancing field of microbiology and is bringing insights into the complexity and adaptations of bacteria causing chronic infection in the respiratory tract. AREAS OF AGREEMENT AND CONTROVERSY: The epidemiology of some infections in CF has changed, with reduction in spread of Burkholderia cenocepacia following patient segregation. Conversely, epidemic strains of Pseudomonas aeruginosa have emerged, which spread between patients; previously, most P. aeruginosa strains were patient-specific. Studies on hypermutators, quorum sensing, biofilm growth and the development of molecular identification have shed light on pathogenicity, microbial adaptation to the host and complexity of infection in CF. Non-tuberculous mycobacteria are emerging pathogens in CF; however, there is much to learn about pathogenicity and treatment of these infections. Species of aerobic and anaerobic bacteria, more commonly encountered in the upper tract, are found in significant numbers in CF sputum. The significance of this is however under debate. Finally, although the clinical relevance of conventional antibiotic susceptibility testing for chronic CF pathogens has been questioned, there are no clear alternatives. EMERGING AREAS FOR DEVELOPING RESEARCH: Much has been learnt about pathogenicity, evolution of CF pathogens and development of antibiotic resistance. The need is to focus on clinical relevance of these observations to improve diagnosis, prevention and treatment of CF infection.

Pyrexia after cardiac surgery: natural history and association with infection.

BACKGROUND: Pyrexia is common after major surgery, and infection is often an important consideration. To investigate the natural history and association with infection, we performed a prospective observational study. METHODS: From November 2000 to January 2001, we studied 219 patients undergoing cardiac surgery screening daily for wound, respiratory, urinary tract, and other infections. Pyrexia was defined as temperature above 37.5 degrees C. RESULTS: Of 219 patients, 7 intraoperative deaths occurred and 1 patient was excluded because of preoperative endocarditis, leaving 211. The mean age (SD) was 64 (10) years, consisting of 172 male patients (81.5%). The proportion pyrexial on days 1, 2, and 5 was 30.0%, 25.8%, and 10.3%, respectively. More patients undergoing urgent or emergency procedures (17.7% versus 7.8%; P =.03) subsequently developed pyrexia. However, there were no differences in wound infection (3.4% versus 8.3%; P =.13), positive cultures for respiratory (14.7% versus 11.4%; P =.16), urinary tract (5.2% versus 2.0%; P =.09), or other infection (8.6% versus 7.3%; P =.71) in patients experiencing postoperative pyrexia compared with those who did not. CONCLUSIONS: Pyrexia is common after cardiac surgery and resolves in the majority of patients by day 5. Because there is no association between early pyrexia and infection, diagnosis of early postoperative infection by pyrexia alone is insufficient and is better established by clinical assessment with microbiological evidence.

European Cystic Fibrosis Society Standards of Care: Framework for the Cystic Fibrosis Centre.

A significant increase in life expectancy in successive birth cohorts of people with cystic fibrosis (CF) is a result of more effective treatment for the disease. It is also now widely recognized that outcomes for patients cared for in specialist CF Centres are better than for those who are not. Key to the effectiveness of the specialist CF Centre is the multidisciplinary team (MDT), which should include consultants, clinical nurse specialist, microbiologist, physiotherapist, dietitian, pharmacist, clinical psychologist, social worker, clinical geneticist and allied healthcare professionals, all of whom should be experienced in CF care. Members of the MDT are also expected to keep up to date with developments in CF through continued professional development, attendance at conferences, auditing and involvement in research. Specialists CF Centres should also network with other Centres both nationally and internationally, and feed Centre data to registries in order to further the understanding of the disease. This paper provides a framework for the specialist CF Centre, including the organisation of the Centre and the individual roles of MDT members, as well as highlighting the value of CF organisations and disease registries.

Mediastinal abscess after lung transplantation secondary to Burkholderia gladioli infection.

Burkholderia gladioli is an unusual organism that has become increasingly responsible for infections in patients who are immunosuppressed, including patients who have undergone solid organ transplantation. This article presents a patient in whom a mediastinal mass due to Burkholderia gladioli developed after lung transplantation. A review of the literature is also presented.

Hypermutability in environmental Pseudomonas aeruginosa and in populations causing pulmonary infection in individuals with cystic fibrosis.

Pseudomonas aeruginosa is the pathogen most commonly associated with morbidity and mortality in cystic fibrosis (CF) patients. The host-pathogen interactions responsible for progressive CF lung diseases are complex. However, there is growing interest in the role of hypermutable P. aeruginosa (that is, those strains with an increased mutation frequency due to mutations in mismatch repair and error prevention genes), in terms of both bacterial adaptation and antimicrobial resistance. The prevalence of hypermutable P. aeruginosa in chronic CF infection has been established, and at 37 % is surprisingly high. To the authors' knowledge, there are no reports of prevalence during the early stages of infection, in environmental pseudomonas, which are believed to be the primary source of infection, and in epidemic strains, which have emerged as a major challenge. The aim of this study was to establish the prevalence of hypermutable P. aeruginosa in these pseudomonas populations. The hypothesis was that hypermutability would be rare in early and in environmental P. aeruginosa but in contrast would explain the relatively recent emergence of epidemic strains. It was found that 10/100 (10 %) of early isolates were strong or weak mutators, suggesting that the CF lung is not the only factor influencing the existence of mutators in this group of patients. Two weak mutators (6 %) were found in 32 environmental isolates. Only two of 15 (13 %) epidemic P. aeruginosa strains were hypermutable, and although closer analysis revealed this issue to be complex, on the whole the data suggested that the atypical characteristics of these highly transmissible strains cannot solely be explained by this phenomenon. The higher than predicted prevalence of mutators in early infection, and in environmental isolates, reinforces the importance of early and aggressive treatment for P. aeruginosa infection in CF.

Allogenic blood transfusion does not predispose to infection after cardiac surgery.

BACKGROUND: Many retrospective studies report increased postoperative infection after allogenic blood transfusion. To investigate this phenomenon, we prospectively studied 232 patients undergoing cardiac surgery. METHODS: Patients were screened daily for evidence of culture positive infections. Wounds were examined daily and defined on the ASEPSIS score. Chest radiographs and white cell counts and differentials were recorded on days 1, 2, and 4. The use of blood products was monitored blindly and independently. Patients were grouped according to transfusion status and compared using chi2 or Fisher's test. Logistic regression analyses were performed to identify predictors of transfusion and infection. RESULTS: Of 232 patients, 116 (50%) received blood product transfusion. Patients receiving blood had lower preoperative hemoglobin, were older, with a greater proportion of urgent/emergency or revision surgery, and were higher risk. Despite this, there were no differences in the frequency of chest infection (20% versus 15%, p = 0.38), urinary infection (3.5% versus 5.3%, p = 0 0.75), wound infection (3.5% versus 8.0%, p = 0.16), or overall infection (28% versus 30%, p = 0.89) comparing the transfused versus untransfused groups. There was no evidence to suggest that administration of blood products was associated with infection (odds ratio 0.92, p = 0.77). CONCLUSIONS: The administration of blood per se did not lead to increased postoperative infection. Clinicians should reconsider withholding blood transfusion in patients solely owing to concerns of predisposition to infection.

Control of microbial contamination during surgical harvest of pig renal xenografts.

The pig has been identified as the most likely source of xenograft material for clinical use and studies are ongoing to overcome the immunological hurdles of pig-to-human transplantation. Attention is now being focussed on identifying and reducing the potential microbiological hazards associated with this technique. Studies have primarily addressed issues surrounding the production and health monitoring of xenograft source pigs and none have so far specifically evaluated the possible risks of microbial contamination during xenograft harvest. In this report, we evaluate the possible routes for contamination of a pig kidney xenograft during organ harvest and describe approaches to the control of these hazards, including the novel use of a custom designed airtight surgical canopy. A standard procedure for microbiological monitoring during xenograft harvest was devised and evaluated. This allowed the rapid identification and anti-microbial sensitivity testing of any isolated organisms. This would enable an early and appropriate pre-emptive treatment of infection because of transmission of pig micro-organisms.