Pseudomonas aeruginosa population genomics among adults with bronchiectasis across Germany

Pseudomonas aeruginosa is a metabolically versatile, aquatic γ-proteobacterium that colonises animate surfaces of plants, animals and humans [1]. During recent decades, this cosmopolitan Gram-negative bacterium has become one of the most frequent causative agents of nosocomial infections, associated with substantial morbidity and mortality [1]. Chronic airway infections with P. aeruginosa are a major cause of morbidity in people with cystic fibrosis (CF), COPD or bronchiectasis.

Short abstract

Genome sequencing of 130 Pseudomonas aeruginosa isolates from 110 bronchiectasis patients identified a few dominant clones common in the global bacterial population and numerous rare clones infrequently seen in the environment or other human infections https://bit.ly/3lIfD2X

To the Editor:

Pseudomonas aeruginosa is a metabolically versatile, aquatic γ-proteobacterium that colonises animate surfaces of plants, animals and humans [1]. During recent decades, this cosmopolitan Gram-negative bacterium has become one of the most frequent causative agents of nosocomial infections, associated with substantial morbidity and mortality [1]. Chronic airway infections with P. aeruginosa are a major cause of morbidity in people with cystic fibrosis (CF), COPD or bronchiectasis.

P. aeruginosa is one of the most frequently isolated pathogens in patients with non-CF bronchiectasis [2]. Clinical studies have revealed that chronic airway infection with P. aeruginosa is associated with more lung lobe involvement and a higher frequency of exacerbations [3, 4]. P. aeruginosa-positive patients had a longer disease course, faster decline of lung function and more severe Bronchiectasis Severity Index stage than those without P. aeruginosa [5, 6, 7]. Patient-to-patient transmission of P. aeruginosa is known to happen frequently in people with CF [8] but molecular epidemiology studies did not substantiate any nosocomial spread among patients with bronchiectasis [9, 10].

Transmission risk is low within families and within bronchiectasis clinics [8]. Thus, most people with bronchiectasis should have acquired P. aeruginosa from other sources, most likely from the inanimate environment. P. aeruginosa is ubiquitously present in aquatic habitats [1]. All organisms, including humans, are exposed to this bacterium, albeit usually far below the threshold of an infectious dose and culture-based microbiology diagnostics [1]. Conversely, P. aeruginosa is isolated from 20% [6] to 33% [11] of respiratory secretions of people attending a bronchiectasis clinic. Considering the high prevalence of P. aeruginosa in people with bronchiectasis, it is clinically relevant whether P. aeruginosa is generally prone to take residence in a bronchiectasis lung or whether a subset of peculiarly adapted clonal lineages preferentially conquers this habitat.

To address this issue, the molecular epidemiology of P. aeruginosa isolates was examined from patients with non-CF bronchiectasis. 296 isolates were collected from March 2018 to November 2020 at the Adult Bronchiectasis Clinic of Hannover Medical School from all patients who had signed the broad consent of the German Center of Lung Research and whose respiratory specimen (sputum as well as nasal and bronchoalveolar lavage) had been P. aeruginosa positive by culture-dependent diagnostics. The cohort consisted of, in total, 110 patients from all over Germany (figure 1a). Overall, 66% of the 110 patients were female (n=72). Mean±sd age was 55±17 years (median 58 years, range 21–83 years), while forced expiratory volume in 1 s was 63±25% of predicted (median 65%, range 17–131% of predicted). The most common aetiologies (in >5% of subjects) were primary ciliary dyskinesia and/or Kartagener syndrome in 32% (n=35), followed by “idiopathic” in 28% (n=31), asthma/allergic bronchopulmonary aspergillosis in 16% (n=17), “other” in 14% (n=15) and nontuberculous mycobacterial pulmonary as well as autoimmune disease in 6% of patients (n=6) each. The 296 P. aeruginosa isolates were analysed by shotgun whole-genome sequencing. The multilocus sequence type (ST) [12] was determined using a mapping-based approach leveraging Illumina short reads against the P. aeruginosa PA14 reference genome. If serial isolates had been collected from one patient, one strain per clone was selected for further analysis. As shown in the phylogram (figure 1b), this set of 130 independent isolates demonstrated a genomically highly diverse P. aeruginosa population at our Adult Bronchiectasis Clinic. In total, 73 STs were identified in the 130 genomes, 53 STs of which were only observed in a single strain (figure 1c). 12 STs were novel and had not been reported before. New STs were assigned by the Pub-MLST database [12]. Of shared STs, we detected four pairs, eight trios and four quartets. Four STs were represented by five or more strains.

FIGURE 1.

Epidemiology of the Pseudomonas aeruginosa population in non-cystic fibrosis (CF) bronchiectasis across Germany compared with that of two international Pseudomonas genome databases. a) Distribution of patients’ place of residence (yellow dot) across Germany, in relation to the Adult Bronchiectasis Clinic at Hannover Medical School (MHH) (red arrow; mean distance 120 km). b) Phylogram of the sequence types (STs) of the 5098 P. aeruginosa genomes in the Pathosystems Resource Integration Center (PATRIC) database. Numerical ST and colour indicate the four most frequent STs identified in the bronchiectasis cohort. c) Clonal distribution of in total 130 P. aeruginosa isolates from people with non-CF bronchiectasis across Germany. Absolute counts of different STs verified by multilocus sequence typing (MLST) are shown. MLST is based on sequence variants in eight highly conserved housekeeping genes [12]. d) Ranks of the 20 most common STs in people with non-CF bronchiectasis in Germany at MHH compared with the top 20 ranks of STs of P. aeruginosa genomes and their absolute numbers (right side in each ST box) deposited in the PATRIC [13, 14], the International Pseudomonas Genome Database (IPGD) [15] and the top 20 ranks of database subgroups therein differentiated by habitat of origin. The chosen categories were (from left to right) chronic human infections, human infections, the hospital environment and the inanimate environment untouched by medicinal activity. Entries from the IPGD [15] were curated for independent isolates if metadata were available. Metadata were unavailable for 2333 out of 4264 genomes deposited with a MLST. The four most common P. aeruginosa STs in people with bronchiectasis are depicted by the same colours in parts b–d.

Figure 1d compares the relative abundance of P. aeruginosa STs among our bronchiectasis population with that of isolates from chronic and acute human infections, the hospital environment, soil and aquatic habitats in the inanimate environment, and the global population documented in the Pathosystems Resource Integration Center [13, 14] and the International Pseudomonas Genome Database [15]. The four most frequent STs found in bronchiectasis airways belonged to the 20 most common clones in the worldwide P. aeruginosa population that are widespread in human infections and in the non-human inanimate environment [12, 15]. Of these four most common clonal lineages in the bronchiectasis isolates, ST27 is characterised by extreme genomic homogeneity, i.e. >90% of strains in the phylogenetic subgroup belong to ST27 [13, 14]. ST17 and ST253 represent the global clones C and PA14 that had been detected in a wide range of habitats all over the world. ST395 represented the most frequently detected clone among our bronchiectasis population. This clonal lineage is less frequent in human infections and rare in the inanimate environment. Thus, ST395 was the only clone in our population that potentially may be overrepresented in bronchiectasis compared to other habitats. However, this assumption needs to be validated in independent bronchiectasis cohorts [16].

P. aeruginosa is one of the bacteria belonging to the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) pathogens that are a threat all over the world due to their capacity to become increasingly resistant to all available antibiotics [1]. Driven by this threat, multiple isolates of the multidrug-resistant high-risk clones ST111, ST146, ST235 and ST274 have been sequenced [12, 13, 15]. Thus, their high frequency in the genome databases may be attributable to clinical needs and focus on hospitals, particularly intensive care units. However, in the context of our Adult Bronchiectasis Clinic, it is reassuring that high-risk clones were only identified at comparably very low frequency.

In summary, our findings indicate that the population biology of P. aeruginosa in bronchiectasis is characterised by a broad range of rare clones and few dominant clonal lineages, which show worldwide distribution in disease and environment. Neither within-centre nosocomial transmission, epidemic virulent clones nor outgrowth lineages that are specific for other diseases were observed. These findings among people with bronchiectasis are in contrast to the long-standing experience in CF where within-family, within-centre, domestic and international spread of epidemic clones has been frequently reported [8], and support the originality of bronchiectasis as a distinct disease entity [4].