Cross-sectional and longitudinal multilocus sequence typing of pseudomonas aeruginosa in cystic fibrosis sputum samples.

Multilocus sequence typing (MLST) is a genetic typing tool designed to provide information about the relatedness of isolates at the core genome level. The utility of MLST in regard to cystic fibrosis (CF)-related infection with Pseudomonas aeruginosa is unknown. The molecular clock speed of the MLST genes was studied using 219 colonies isolated longitudinally from 49 patients with CF. A cross-sectional study examining 27 to 46 colonies per sputum sample for samples from 16 patients was also undertaken. The molecular clock speed was estimated to be 2.05 x 10(-5) (upper 95% confidence limit) or 4.75 x 10(-6) (50% confidence limit) point mutations per nucleotide per year. In the cross-sectional study, 50% of patients were infected with more than one sequence type. There was evidence of point mutations, recombination events, and coinfection with epidemic and unique strains. A clonal complex that was highly genetically distinct from the rest of the P. aeruginosa population was identified. The MLST scheme uses genes with an appropriate clock speed and provides useful information about the genetic variation of P. aeruginosa within and between patients with CF.

Association between hypermutator phenotype, clinical variables, mucoid phenotype, and antimicrobial resistance in Pseudomonas aeruginosa.

The presence of hypermutator Pseudomonas aeruginosa was associated with poorer lung function in patients at the Adult West Midlands CF Unit. Mucoid isolates were more likely to be hypermutators. The presence of resistant mutant subpopulations was associated with hypermutator phenotype but was not good enough to be used as a test for this phenotype.

Elucidating global epidemiology of Burkholderia multivorans in cases of cystic fibrosis by multilocus sequence typing.

Burkholderia multivorans is a prominent B. cepacia complex (BCC) species causing infection in people with cystic fibrosis. Despite infection control measures being introduced to reduce the spread of BCC there is a continued emergence of infections by B. multivorans. Our objective was to analyze a global collection of B. multivorans isolates, comparing those from environmental and clinical sources with those from reported outbreaks. Multilocus sequence typing (MLST) was performed on 107 B. multivorans isolates to provide a detailed analysis of the global population biology of this species. MLST resolved 64 B. multivorans sequence types. Twelve of these were globally distributed and associated with human infection; two of these (ST-21 and ST-375) were also composed of environmental isolates. These global lineages included strains previously linked to large outbreaks (e.g., French epidemic clone ST-16). Though few environmental isolates of B. multivorans were available for analysis, of six strains identified, three were identical to strains recovered from cystic fibrosis (CF) infection. Although the ability of B. multivorans to cause CF outbreaks is known, our report here concerning the existence of globally distributed B. multivorans CF strains is a new observation for this emerging B. cepacia complex pathogen and suggests that certain strain types may be better adapted to human infection than others. Common transmission-associated risk factors were not obviously linked to the globally distributed strains; however, the overlap in strains recovered from water environments, industrial products, and human infection suggests that environmental sources may be an important reservoir for infection with B. multivorans.

Cross-infection in cystic fibrosis: the knowledge and behaviour of adult patients.

INTRODUCTION: The knowledge and behaviour of adult patients with cystic fibrosis (CF) regarding cross-infection are ill understood. METHODS: A questionnaire was designed to investigate this at the West Midlands Adult CF Centre. RESULTS: 94 patients completed the questionnaire. 54%, 36% and 46% had "no idea" of the lifetime risk of contracting Burkholderia cepacia complex, epidemic strains of Pseudomonas aeruginosa, and MRSA, respectively. 25-33% did not know the consequences of infection with these bacteria. 35% mixed with other people with CF, 6.5% during physiotherapy or nebulizer use. Most respondents did not think quality of life was significantly linked with segregation from other patients with CF. CONCLUSIONS: Adults with CF, at least in the West Midlands, have poor knowledge of the risk and consequences of cross-infection. A significant proportion ignored advice not to mix with other patients, although segregation was not thought to impact upon quality of life. This suggests that more education about the risks of cross-infection would be beneficial.

Reliability of multilocus sequence typing of the Burkholderia cepacia complex in cystic fibrosis.

INTRODUCTION: Infection with the Burkholderia cepacia complex is an important cause of morbidity and mortality in cystic fibrosis (CF). We investigated the molecular clock speed of the seven genes used in the multilocus sequence typing (MLST) scheme for these bacteria. METHODS: At least two isolates, separated by months to years, from each of 20 patients were typed using MLST. In total 41 isolates, providing 128 isolate-years, were analyzed. Mutation and recombination rates were estimated assuming a Poisson distribution. RESULTS: Out of 20 patients, 15 had no change in sequence type over time (mean 7.07 years, range 1.09 to 14.24). One patient had strain replacement. Three patients had evidence of recombination involving one of the seven housekeeping genes, and one patient had evidence of recombination of two genes. The mutation rate was estimated as 2.36x10(-6) per nucleotide per year (50% confidence limit) and 1.02x10(-5) per nucleotide per year (upper 95% confidence limit). The rate of nucleotide changes due to recombination events was estimated as 0.676 to 0.839 per year (95% confidence limits). CONCLUSIONS: B. cepacia complex housekeeping genes have a slow molecular clock speed and MLST provides a robust and reliable typing technique for isolates from this complex. A low rate of point mutation was found, with a higher rate of recombination events, in keeping with previous cross-sectional epidemiological data. The study also demonstrated, for the first time, recombination in a longitudinal in vivo study.

Environmental Burkholderia cepacia complex isolates in human infections.

Members of the Burkholderia cepacia complex (Bcc), found in many environments, are associated with clinical infections. Examining diverse species and strains from different environments with multilocus sequence typing, we identified > 20% of 381 clinical isolates as indistinguishable from those in the environment. This finding links the natural environment with the emergence of many Bcc infections.