Genetic Analysis of Pseudomonas aeruginosa Isolates from the Sputa of Australian Adult Cystic Fibrosis Patients

Abstract

Genetic investigations were carried out with 50 phenotypically selected strains of Pseudomonas aeruginosa from 18 patients attending an Australian cystic fibrosis (CF) center. The isolates were analyzed by restriction fragment length polymorphism (RFLP) analysis by pulsed-field gel electrophoresis (PFGE). Phylogenetic analysis of the macrorestriction patterns showed rates of genetic similarity ranging from 76 to 100%; 24 (48%) of the strains from 11 patients had greater than 90% similarity. A dominant strain emerged: 15 isolates from seven patients had identical PFGE patterns, and 4 other isolates were very closely related. The 50 isolates were grouped into 21 pulsotypes on the basis of visual delineation of a three-band difference. Ten of the 18 (56%) patients were infected with clonal or subclonal strains. Sequence analysis of PCR products derived from the mucA gene showed 20 mutations, with the number of mutations in individual isolates ranging from 1 to 4; 19 of these changes are reported here for the first time. Potentially functional changes were found in 22 (44%) isolates. Eight changes (five transversions and three single base deletions) led to premature stop codons, providing support for the presence of mucA mutations as one pathway to mucoidy. There was a trend toward an association between the dominant strain and lack of potentially functional mucA mutations (P ‫؍‬ 0.09 by the 2 test) but no relationship between genotype and phenotype. This is the first study of genetic variation in P. aeruginosa isolates from adult Australian CF patients. The findings highlight the need for further investigations on the transmissibility of P. aeruginosa in CF patients.Chronic lung infection with Pseudomonas aeruginosa leading to respiratory failure is the major cause of both morbidity and mortality in patients with cystic fibrosis (CF) (6). Genetic analyses by techniques such as restriction fragment length polymorphism (RFLP) analysis by pulsed-field gel electrophoresis (PFGE) have indicated that most CF patients are colonized for long periods with single clones or subclones of P. aeruginosa (4,7,9,17,21,23), and the appearance of new unrelated strains has often been associated with changes in antibiotic therapy (3). It has been believed that P. aeruginosa is generally environmentally acquired and that person-to-person spread occurs only rarely (3,5,9,11,16,20,21,23). However, recent reports of the transmission of virulent multidrug-resistant mucoid strains of P. aeruginosa in a large Australian pediatric CF clinic (1) and two adult CF clinics in the United Kingdom (10, 13) and the person-to-person spread of P. aeruginosa among holiday campers with CF in Germany (9) have raised concerns about the effectiveness of standard infection control practices for P. aeruginosa. Nonetheless, confirmation of substantial cross infection with epidemic strains would be needed to support a policy of segregation like the one already practiced for Burkholderia cepacia.Associated with the chronic colonization of P. aeruginosa in CF pat...

Investigation of the algT operon sequence in mucoid and non-mucoid Pseudomonas aeruginosa isolates from 115 Scandinavian patients with cystic fibrosis and in 88 in vitro non-mucoid revertants

“…However, the same types of mutations were also found in unrelated clones from different geographical locations in Scandinavia, suggesting that these loci represent hot-spot regions for mutation in mucA. In addition, the mucA22 mutation was reported previously in several mucoid isolates from the USA, Europe and Australia (Boucher et al, 1997b;Bragonzi et al, 2006;Anthony et al, 2002). Isolates belonging to the Dk1, Dk2 and N clones showed several other types of mutations.…”

Airway epithelial control of Pseudomonas aeruginosa infection in cystic fibrosis

“…When mutations arise in the mucA gene leading to loss of intact protein production, the AlgU protein is made available to activate transcription of the alg locus and enhance alginate production. Although reports indicate that there is a common mutation or hot spot in the mucA gene that leads to early termination of translation, a mutation that was found in >84% of clinical mucoid isolates in two studies [48,50], analysis of isolates in Australia showed that a lower percentage (44%) of mucoid strains had this genetic change [51]. Other genetic regulators and P. aeruginosa proteins have also been reported to not only affect alginate production but also other potential virulence factors in a coordinated fashion.…”

Factors associated with infection by Pseudomonas aeruginosa in adult cystic fibrosis

“…The Manchester genotype does not appear to induce an exaggerated inflammatory response in its host despite increased treatment requirements in these patients [19]. In an Australian study, eight out of 18 patients were found to be infected with a clustered P. aeruginosa genotype, and this strain lacked the functional mucA mutations responsible for over-production of alginate and mucoidy in the unique strains [20]. In other words, alternative mechanisms must have been operational in the clustered strain to lead to mucoidy [20].…”

“…In an Australian study, eight out of 18 patients were found to be infected with a clustered P. aeruginosa genotype, and this strain lacked the functional mucA mutations responsible for over-production of alginate and mucoidy in the unique strains [20]. In other words, alternative mechanisms must have been operational in the clustered strain to lead to mucoidy [20]. Multiple and diverse bacterial factors associated with the ability of cross-infecting P. aeruginosa strains to successfully establish persistent infection in other hosts are conceivable including: an improved ability to aerosolise [9]; a propensity to infect sheltered extrapulmonary sites [21] or adhere to CF mucosal surfaces [22]; a better ability to prosper in the abnormally anaerobic environment of the airway surface liquid typical of older CF patients; an increased tendency to form biofilms in the respiratory tract; or increased intrinsic antibiotic resistance [3] (in turn related to the propensity to form biofilms) [23].…”