D. Kenna

Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care.

Clusters of genetically similar isolates of Pseudomonas aeruginosa from multiple hospitals in the UK

Variable number tandem repeat (VNTR) analysis at nine loci of isolates of Pseudomonas aeruginosa submitted to the national reference laboratory from UK hospitals, from over 2000 patients, between June 2010 and June 2012 revealed four widely found types that collectively were received from approximately a fifth of patients, including from those with cystic fibrosis. These types were also prevalent among related submissions from the clinical environment and were received from up to 54 (out of 143) hospitals. Multi-locus sequence typing and blaOXA-50-like sequencing confirmed the clonal relationship within each cluster, and representatives from multiple centres clustered within about 70 % by pulsed-field gel electrophoresis. Illumina sequencing of 12 isolates of cluster A of VNTR profile 8, 3, 4, 5, 2, 3, 5, 2, x (where the repeat number at the last, most discriminatory locus is variable) revealed a large number of variably present targets in the accessory genome and seven of these were sought by PCR among a larger set of isolates. Representatives from patients within a single centre mostly had distinct accessory gene profiles, suggesting that these patients acquired the strain independently, while those with clear epidemiological links shared the same profile. Profiles also varied between representatives from different centres. Epidemiological investigations of widely found types such as these require the use of finer-typing methods, which increasingly will be informed by next generation sequencing.

Molecular Fingerprinting of Mycobacterium abscessus Strains in a Cohort of Pediatric Cystic Fibrosis Patients

ABSTRACT Forty-one Mycobacterium abscessus complex isolates from 17 pediatric cystic fibrosis (CF) patients were typed using a novel variable-number tandem repeat (VNTR) scheme and an automated repetitive-PCR (rep-PCR) system. Both VNTR and rep-PCR typing methods differentiate between members of the M. abscessus complex. The isolates from individual patients are indistinguishable, and the data strongly suggest that individual CF patients are persistently infected with one strain and also suggests that different CF patients can harbor the same strain.

Whole-Genome Sequencing and Epidemiological Analysis Do Not Provide Evidence for Cross-transmission of Mycobacterium abscessus in a Cohort of Pediatric Cystic Fibrosis Patients

We have not been able to demonstrate cross-transmission of Mycobacterium abscessus within our hospital, except between siblings who had intense contact in the home environment. The role of the environment in the acquisition of M. abscessus infection requires further investigation.

A novel culture medium for isolation of rapidly-growing mycobacteria from the sputum of patients with cystic fibrosis

BACKGROUND Isolation of mycobacteria from the sputum of patients with cystic fibrosis (CF) is challenging due to the overgrowth of cultures by other bacteria and fungi. In this setting, Burkholderia cepacia selective agar (BCSA) has been recommended as a convenient and effective culture medium for the isolation of rapidly-growing, non-tuberculous mycobacteria (NTM). A novel selective culture medium (RGM medium) was evaluated for the isolation of rapidly-growing NTM from the sputum of children and adults with CF. METHODS A total of 118 isolates of rapidly-growing mycobacteria and 98 other bacteria and fungi were inoculated onto RGM medium. These were assessed for growth at 30°C over a seven day period. A total of 502 consecutive sputum samples were collected from 210 patients with CF. Each sample was homogenized and cultured onto RGM medium and also onto BCSA. Cultures were incubated for 10days at 30°C. RESULTS Of 118 isolates of mycobacteria all but one grew well on RGM medium, whereas 94% of other bacteria and fungi were inhibited. A total of 55 sputum samples (from 33 distinct patients) yielded NTM using a combination of both RGM and BCSA (prevalence: 15.7%). NTM were recovered from 54 sputum samples using RGM medium compared with only 17 samples using BCSA (sensitivity 98% vs. 31%; P≤0.0001). A total of 419 isolates of non-mycobacteria were recovered from sputum samples on BCSA compared with 46 on RGM medium. CONCLUSIONS RGM medium offers a simple and effective culture method for the isolation of rapidly-growing mycobacteria from sputum samples from patients with CF without decontamination of samples. RGM medium allows for the systematic screening of all sputum samples routinely referred for culture from patients with CF.

Molecular epidemiological analysis suggests cross-infection with Pseudomonas aeruginosa is rare in non-cystic fibrosis bronchiectasis

To the Editor: In both cystic fibrosis (CF) and non-CF bronchiectasis (NCFBr) chronic Pseudomonas aeruginosa infection is adversely prognostic [1, 2]. In CF, epidemic infections with specific clones of P. aeruginosa are associated with further adverse outcomes [3, 4]. This cross-infection risk has led to segregation of patients [5]. There are few data on P. aeruginosa cross-infection in NCFBr. As a result, segregation in NCFBr has not been addressed in guidelines [6]. Our aim was to undertake a cross-infection study in NCFBr. This was undertaken in an adult bronchiectasis service in the north-east of England (UK) that is separated from the regional CF unit (sited 2 miles (3 km) away). The service was initiated in 2007 with a weekly specialist clinic without a Pseudomonas -specific clinic. When NCFBr patients are hospitalised, there is a preference for cubicle-based (single-patient room) management, but when cubicles are unavailable, patients are managed in six-bed bays. All patients had computed tomographic confirmation and had predominantly idiopathic or post-infectious bronchiectasis with CF excluded following current guidelines [6]. The study had ethical permission and Caldicott approval (Newcastle and North Tyneside National Research Ethics Service Committee). 56 isolates were selected for analysis. Six were chosen from CF patients as laboratory controls. 50 were NCFBr isolates collected between 2008 and 2011 from …

Use of nrdA gene sequence clustering to estimate the prevalence of different Achromobacter species among Cystic Fibrosis patients in the UK

BACKGROUND We aimed to estimate the prevalence of different Achromobacter species among UK Cystic Fibrosis (CF) patients. METHODS nrdA sequence clustering was used to identify 147 Achromobacter isolates from 96 patients from 27 hospitals to species level. Potential cross-infection was investigated by MLST, pulsed-field gel electrophoresis and whole genome sequencing (WGS). RESULTS Achromobacter xylosoxidans was the most prevalent species affecting 59 of 96 (61%) patients, followed by Achromobacter insuavis and Achromobacter dolens (12.4% and 8%, respectively). Three novel nrdA clusters were identified. One was further characterised by sequencing the intrinsic blaOXA gene, revealing novel variants. WGS of A. insuavis 2a isolates from four patients attending the same paediatric unit revealed that three were ST144, but differed from one another by a minimum of 385 SNPs, suggesting cross-infection was unlikely. CONCLUSIONS nrdA sequence clustering permitted an estimation of UK Achromobacter species prevalence, highlighted additional novel species, and aided cross-infection investigations.

Mycobacterium abscessus infection in cystic fibrosis: molecular typing and clinical outcomes.

Mycobacterium abscessus is a significant pathogen in the cystic fibrosis patient population. PCR amplification and sequencing can provide accurate subspecies identification, and can predict macrolide susceptibility, which is becoming increasingly important for patient management. Molecular techniques for further typing of isolates provide tools for the ongoing investigations into the clinical impact of particular M. abscessus strains. Whole-genome sequencing is likely to be the only technique that provides sufficient resolution for investigating transmission events between patients.

Burkholderia latens infection in cystic fibrosis

As our knowledge and understanding of organisms of the Burkholderia cepacia complex (Bcc) progresses, further species have been recognised that are of (as yet) uncertain clinical significance [1]. With this letter we report on three patients attending our adult CF unit who are all chronically infected with Burkholderia latens, a species of Bcc that was only described in 2008, and for which there is a paucity of clinical outcome data. Moreover, all three patients have lived within a narrow geographical radius, which raises the possibilities of either a common environmental source or transmissibility of B. latens between patients. It is too early to infer the longer term consequences of B. latens infection, but so far none of the patients appear to have suffered a deterioration attributable to the infection.

Prevalence of Burkholderia species, including members of Burkholderia cepacia complex, among UK cystic and non-cystic fibrosis patients

Purpose. We aimed to establish the prevalence of different Burkholderia species among UK cystic fibrosis (CF) and non‐CF patients over a 2 year period. Methodology. Matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry was used to identify isolates to genus level, followed by recA/gyrB sequence clustering or species‐specific PCR. In all, 1047 Burkholderia isolates were submitted for identification from 361 CF patients and 112 non‐CF patients, 25 from the hospital environment and three from a commercial company. Potential cross‐infection was assessed by pulsed‐field gel electrophoresis (PFGE) and multi‐ locus‐sequence typing (MLST). MICs were determined for 161 Burkholderia cepacia complex (Bcc) isolates. CF Trust registry data were sought to examine clinical parameters relating to Bcc infection. Results. Burkholderia multivorans was the most prevalent species among CF patients affecting 56% (192) patients, followed by Burkholderia cenocepacia IIIA (15%; 52 patients). Five novel recA clusters were found. Among non‐CF patients, Burkholderia cepacia was the most prevalent species (37/112; 34%), with 18 of 40 isolates part of a UK‐wide B. cepacia ‘cluster’. This and three other clusters were investigated by PFGE and MLST. Cable‐pili positive isolates included two novel sequence types and representatives of ET12. Antibiotic susceptibility varied between and within species and CF/non‐ CF isolates. CF Trust registry data suggested no significant difference in lung function between patients harbouring B. cenocepacia, B. multivorans and other Bcc species (P=0.81). Conclusion. The dominance of B. multivorans in CF, the presence of a B. cepacia cluster among non‐CF patients and the existence of putative novel species all highlighted the continuing role of Burkholderia species as opportunistic pathogens.