Pseudomonas aeruginosa Strain-sharing in Early Infection Among Children With Cystic Fibrosis

Abstract

Background: We previously identified Pseudomonas aeruginosa isolates with characteristics typical of chronic infection in some early infections in children with cystic fibrosis (CF), suggesting that these isolates may have been acquired from other patients. Our objective was to define the extent of P. aeruginosa strain-sharing in early CF infections and its impact on antibiotic eradication treatment failure rates.

Methods: We performed whole genome sequencing on isolates from early pediatric CF pulmonary infections and from the following comparator groups in the same hospital: chronic CF infection, sink drains, sterile site infections, and asymptomatic carriage. Univariate logistic regression was used to assess factors associated with treatment failure.

Results: In this retrospective, observational study, 1029 isolates were sequenced. The CF clones strain B and clone C were present. In 70 CF patients with early infections, 14 shared strains infected 29 (41%) patients over 5 years; 16% (n = 14) of infections had mixed strains. In the 70 children, approximately one-third of shared-strain infections were likely due to patient-to-patient transmission. Mixed-strain infections were associated with strain-sharing (odds ratio, 8.50; 95% confidence interval, 2.2-33.4; P = .002). Strain-sharing was not associated with antibiotic eradication treatment failure; however, nosocomial strain transmission was associated with establishment of chronic infection in a CF sibling pair.

Conclusions: Although early P. aeruginosa CF infection is thought to reflect acquisition of diverse strains from community reservoirs, we identified frequent early CF strain-sharing that was associated with the presence of mixed strains and instances of possible patient-to-patient transmission.

Keywords: Pseudomonas aeruginosa; cross infection; cystic fibrosis; respiratory tract infections; whole genome sequencing.

Figure 1.

A, Unrooted neighbour-joining mashtree of 1029 de novo assemblies from the Hospital for Sick Children Pseudomonas aeruginosa (Pa) isolates and 81 complete Pa reference genomes. Most sequences cluster in either group 1, which contains reference strain pa01, or group 2, which contains reference strain pa14. This population structure is similar to those from previous reports of phylogenetic analyses of diverse Pa isolates. The remaining outlier sequences cluster in group 3 together with the pa7 reference genome. Note that pa7 is a phylogenetic outlier that diverged early in evolutionary history from other Pa lineages. Note that a branch leading to a sterile site isolate (STS031) in group 3 is truncated. B, Neighbour-joining tree from panel A with the group 3 branch shown to scale. C, A circular mashtree cladogram (branch lengths ignored). Isolates from the different cohorts (CF and non-CF patients and hospital sinks) are dispersed across the tree. A group of closely related sequences from the early and chronic cohorts is circled and shown in an enlarged phylogram (branch lengths proportional to evolutionary distance) on the right. Sequences from early cohort case 49 (SK049) and chronic cohort case 14 (HSC014) appear highly related. In fact, some sequences from SK049 appear as closely related to HSC014 sequences as they are to other SK049 sequences. This group was therefore subjected to further analysis by mapping sequencing reads to the most closely related reference genome, in this case PAER_119, to generate a pairwise single-nucleotide polymorphism distance matrix and maximum likelihood (ML) tree and thereby determine if these sequences represent a shared strain. Isolate coding system: early cohort, 594 (isolate number) SK (early cohort) 049 (patient number) m1 (morphotype number) i2 (isolate number) 11 (collection year 2011); chronic cohort, HSC (chronic cohort) 041 (patient number) B2 (visit type and number; A: enrollment, B: baseline, E: exacerbation, F: follow up) -1(isolate number); environmental cohort, ENV (environmental cohort) 64 (room number where isolate collected)-3 (isolate number). Abbreviation: CF, cystic fibrosis.

Figure 2.

Maximum likelihood trees for 11 shared strains in early cohort patients rooted on reference genome (not shown) used for SNP calling as detailed in Supplementary Table 1. Strain 51 contains a long branch to 2 hypermutator isolates from case SK029 (475 and 476). These isolates are 89 to 90 SNPs different from isolates from infections that occurred in 1 case (SK032) in 2013 and 2015. We identified a mutS DNA mismatch repair gene frameshift mutation at codon 333 (of total 2568), resulting in nonfunctional MutS in isolates 475 and 476 only, and so consider them part of the shared strain. The SK032 isolates are paraphyletic with respect to 475 and 476, which suggests the direction of Pseudomonas aeruginosa transmission was from SK032 to SK029. Abbreviation: SNP, single-nucleotide polymorphism.

Figure 3.

A, Maximum likelihood tree of strain 1 isolates rooted on reference genome (not shown) used for SNP calling as detailed in Supplementary Table 1. Isolates of ST17 Pseudomonas aeruginosa (Pa) from 3 early cohort cases (SK020, SK028, and SK055) are identical to each other and to 1 isolate from a chronic cohort case (HSC020). The chronic case exhibits significant intrapatient Pa sequence diversity from the time of enrollment (sample A) through baseline pulmonary status assessments (B), pulmonary exacerbations (E), and follow-up visits postexacerbation (F). However, only 1 HSC020 isolate, B4–1, is identical to the early patient isolates, which allowed us to date the approximate period that strain transmission occurred to when this isolate was collected in September 2012. B, Timeline of Pa infections and hospital visits. Each row represents 1 patient. Dark vertical lines: clinic or pulmonary function test (PFT) laboratory visits. Light gray boxes: inpatient admissions. Dark circles: sputum was positive for Pa and isolates were sequenced, with presence of shared strain confirmed. Light gray circles: sputum positive for Pa, but isolates were not sequenced. Sibling cases SK020 and SK055 were admitted to the cystic fibrosis ward in September 2012 and overlapped for 6 days with the stay of the chronic cohort patient. Shortly thereafter, SK020 and SK055 had new-onset infection with the chronic patient’s strain. Another early cohort patient, SK028, was infected in August 2012, several months after an admission to the ward that commenced on the day after the chronic patient had been discharged. C, Maximum likelihood tree of strain 3 isolates rooted on the reference genome. Isolates from early cohort cases SK006 and SK007 are identical and separated by 4 SNPs from an isolate from SK052 (the sibling of SK007) and an isolate from chronic cohort case HSC032. The siblings developed chronic infection with this Pa strain and were subsequently enrolled in the biofilm trial. All subsequent chronic isolates from the siblings were with the same strain and were sequenced under the study code numbers HSC040 (for SK0007) and HSC035 (for SK052). D, Timeline of Pa infections and hospital visits as in (B). Each row represents 1 patient (SK007 and SK052 also have chronic cohort study codes HSC035 and HSC040). SK006 attended the clinic in July 2011 and had a new-onset infection detected. On the same day, the siblings also attended the clinic; SK007 developed infection with the shared strain 1 month later, while SK052 developed infection with the shared strain in December. SK052 could have acquired the strain from SK006 at the clinic visit or from their sibling subsequently. In May 2012, HSC032 was superinfected with the shared strain but had no overlapping visits with the other patients in the prior 6 months. Afterward, HSC032 reverted to their preexisting Pa strain. Abbreviation: SNP, single-nucleotide polymorphism.

Figure 4.

A, Maximum likelihood tree of isolates rooted on the reference genome (not shown) used for SNP calling as detailed in Supplementary Table 1. One isolate from chronic case HSC021 (HSC021-A1–1, collected at trial enrollment in 2010) is 3 SNPs from isolates from a sink in cystic fibrosis (CF) ward room 64 (Env64 1–4). The other 9 isolates from HSC020 (collected in 2010–2012) are 5 to 9 SNPs distant. An isolate from a sterile site infection (STS012) is also 3 SNPs from the sink isolates and 4 SNPs from HSC021-A1–1. Isolates from early cohort case SK005 were included as they appeared closely related to the other isolates on the mashtree. However, we found at least 6 SNP differences from all other isolates, so we deemed the SK005 isolates to be unique, that is, they are not part of the shared strain. B, Floor map of the inpatient CF ward with locations of sink samples and inpatient admissions. Case HSC021 was admitted in May 2011 to a room opposite room 64 where the sink isolates were retrieved 7 years later. This suggests isolates from HSC021 were introduced to the ward environment during the 2011 admission and persisted in the ward sinks. The STS012 isolate came from a postoperative blood culture from a 2001 patient (non-CF) with no epidemiological links to this ward. Abbreviation: Pa, Pseudomonas aeruginosa; PEx, pulmonary exacerbation; SNP, single-nucleotide polymorphism.