Stenotrophomonas maltophilia natural history and evolution in the airways of adults with cystic fibrosis.

Introduction: Stenotrophomonas maltophilia is an opportunistic pathogen infecting persons with cystic fibrosis (pwCF) and portends a worse prognosis. Studies of S. maltophilia infection dynamics have been limited by cohort size and follow-up. We investigated the natural history, transmission potential, and evolution of S. maltophilia in a large Canadian cohort of 321 pwCF over a 37-year period. Methods: One-hundred sixty-two isolates from 74 pwCF (23%) were typed by pulsed-field gel electrophoresis, and shared pulsotypes underwent whole-genome sequencing. Results: S. maltophilia was recovered at least once in 82 pwCF (25.5%). Sixty-four pwCF were infected by unique pulsotypes, but shared pulsotypes were observed between 10 pwCF. In chronic carriage, longer time periods between positive sputum cultures increased the likelihood that subsequent isolates were unrelated. Isolates from individual pwCF were largely clonal, with differences in gene content being the primary source of genetic diversity objectified by gene content differences. Disproportionate progression of CF lung disease was not observed amongst those infected with multiple strains over time (versus a single) or amongst those with shared clones (versus strains only infecting one patient). We did not observe evidence of patient-to-patient transmission despite relatedness between isolates. Twenty-four genes with ≥ 2 mutations accumulated over time were identified across 42 sequenced isolates from all 11 pwCF with ≥ 2 sequenced isolates, suggesting a potential role for these genes in adaptation of S. maltophilia to the CF lung. Discussion: Genomic analyses suggested common, indirect sources as the origins of S. maltophilia infections in the clinic population. The information derived from a genomics-based understanding of the natural history of S. maltophilia infection within CF provides unique insight into its potential for in-host evolution.

The natural history and genetic diversity of Haemophilus influenzae infecting the airways of adults with cystic fibrosis.

Haemophilus influenzae is a Gram-negative pathobiont, frequently recovered from the airways of persons with cystic fibrosis (pwCF). Previous studies of H. influenzae infection dynamics and transmission in CF predominantly used molecular methods, lacking resolution. In this retrospective cohort study, representative yearly H. influenzae isolates from all pwCF attending the Calgary Adult CF Clinic with H. influenzae positive sputum cultures between 2002 and 2016 were typed by pulsed-field gel electrophoresis. Isolates with shared pulsotypes common to ≥ 2 pwCF were sequenced by Illumina MiSeq. Phylogenetic and pangenomic analyses were used to assess genetic relatedness within shared pulsotypes, and epidemiological investigations were performed to assess potential for healthcare associated transmission. H. influenzae infection was observed to be common (33% of patients followed) and dynamic in pwCF. Most infected pwCF exhibited serial infections with new pulsotypes (75% of pwCF with ≥ 2 positive cultures), with up to four distinct pulsotypes identified from individual patients. Prolonged infection by a single pulsotype was only rarely observed. Intra-patient genetic diversity was observed at the single-nucleotide polymorphism and gene content levels. Seven shared pulsotypes encompassing 39% of pwCF with H. influenzae infection were identified, but there was no evidence, within our sampling scheme, of direct patient-to-patient infection transmission.

Staphylococcus aureus in Non-Cystic Fibrosis Bronchiectasis: Prevalence and Genomic Basis of High Inoculum ß-Lactam Resistance.

Rationale: The pathobiology of Staphylococcus aureus in non-cystic fibrosis bronchiectasis (nCFB) is poorly defined. When present at high density or "inoculum," some methicillin-sensitive S. aureus (MSSA) can inefficiently degrade antistaphylococcal ß-lactam antibiotics via BlaZ penicillinases (termed the "inoculum effect" [IE]). Given the high burden of organisms in bronchiectatic airways, this is particularly relevant. Objectives: Drawing from a prospectively collected biobank, we sought to understand the prevalence, natural history, potential for transmission, and antibiotic resistance profiles among nCFB-derived MSSA isolates. Methods: All individuals attending a regional consultancy nCFB clinic with sputum collected between 1981 and 2017 were considered, and those with one or more S. aureus-positive cultures composed the cohort. Each individual's most recent biobank isolate was subjected to whole-genome sequencing (including the blaZ gene), antibacterial susceptibility testing, and comparative ß-lactam testing at standard (5 × 105 colony-forming unit [cfu]/ml) and high (5 × 107 cfu/ml) inocula to assess for the IE and pronounced IE. Results: Seventy-four (35.4%) of 209 individuals had one or more sputum samples with S. aureus (68 MSSA, 6 methicillin-resistant S. aureus). Those with S. aureus infection were more likely to be female. Among 60 of 74 MSSA isolates subjected to whole-genome sequencing, no evidence of transmission was identified, although specific multilocus sequence typing types were prevalent, including ST-1, ST-15, ST-30, and ST-45. Antibiotic resistance was uncommon, except for macrolides (∼20%). Among the 60 MSSA samples, the prevalence of IE and pronounced IE was observed to be drug specific: meropenem (0% and 0%, respectively), cefepime (3% and 5%, respectively), ceftazidime (8% and 0%, respectively), cloxacillin (12% and 0%, respectively), cefazolin (23% and 0%, respectively), and piperacillin-tazobactam (37% and 17%, respectively). The cefazolin IE was associated with blaZ type A (P < 0.01) and ST-30 (P < 0.01), whereas the piperacillin-tazobactam IE was associated with type C blaZ (P < 0.001) and ST-15 (P < 0.05). Conclusions:S. aureus infection was common, although no evidence of transmission was apparent in our nCFB cohort. Although routine susceptibility testing did not identify significant resistance, inoculum-related resistance was found to be relevant for commonly used nCFB antibiotics, including cefazolin and piperacillin-tazobactam. Given previous associations between IEs and negative patient outcomes, further work is warranted to understand how this phenotype impacts nCFB disease progression.

Epidemiology of E. coli in Cystic Fibrosis Airways Demonstrates the Capacity for Persistent Infection but Not Patient-Patient Transmission.

Escherichia coli is frequently isolated from the respiratory secretions of cystic fibrosis (CF) patients yet is not considered a classical CF pathogen. Accordingly, little is known about the natural history of this organism in the CF airways, as well as the potential for patient-to-patient transmission. Patients attending the Calgary Adult CF Clinic (CACFC) between January 1983 and December 2016 with at least one E. coli-positive sputum culture were identified by retrospective review. Annual E. coli isolates from the CACFC biobank from each patient were typed by pulsed-field gel electrophoresis (PFGE) and isolates belonging to shared pulsotypes were sequenced. Single nucleotide polymorphism (SNP) and phylogenetic analysis were used to investigate the natural history of E. coli infection and identify potential transmission events. Forty-five patients with E. coli-positive sputum cultures were identified. Most patients had a single infection episode with a single pulsotype, while replacement of an initial pulsotype with a second was observed in three patients. Twenty-four had E. coli recovered from their sputum more than once and 18 patients had persistent infections (E. coli carriage >6 months with ≥3 positive cultures). Shared pulsotypes corresponded to known extraintestinal pathogenic E. coli strains: ST-131, ST-73, and ST-1193. Phylogenetic relationships and SNP distances among isolates within shared pulsotypes were consistent with independent acquisition of E. coli by individual patients. Most recent common ancestor date estimates of isolates between patients were inconsistent with patient-to-patient transmission. E. coli infection in CF is a dynamic process that appears to be characterized by independent acquisition within our patient population and carriage of unique sets of strains over time by individual patients.

Spatiotemporal modulation of abscisic acid and gibberellin metabolism and signalling mediates the effects of suboptimal and supraoptimal temperatures on seed germination in wheat (Triticum aestivum L.).

Seed germination is a complex process regulated by intrinsic hormonal cues such as abscisic acid (ABA) and gibberellin (GA), and environmental signals including temperature. Using pharmacological, molecular and metabolomics approaches, we show that supraoptimal temperature delays wheat seed germination through maintaining elevated embryonic ABA level via increased expression of ABA biosynthetic genes (TaNCED1 and TaNCED2), increasing embryo ABA sensitivity through upregulation of genes regulating ABA signalling positively (TaPYL5, TaSnRK2, ABI3 and ABI5) and decreasing embryo GA sensitivity via induction of TaRHT1 that regulates GA signalling negatively. Endospermic ABA and GA appeared to have minimal roles in regulating germination at supraoptimal temperature. Germination inhibition by suboptimal temperature is associated with elevated ABA level in the embryo and endosperm tissues, mediated by induction of TaNCEDs and decreased expression of endospermic ABA catabolic genes (TaCYP707As), and increased ABA sensitivity in both tissues via upregulation of TaPYL5, TaSnRK2, ABI3 and ABI5 in the embryo and TaSnRK2 and ABI5 in the endosperm. Furthermore, suboptimal temperature suppresses GA synthesis in both tissues and GA sensitivity in the embryo via repressing GA biosynthetic genes (TaGA20ox and TaGA3ox2) and inducing TaRHT1, respectively. These results highlight that spatiotemporal modulation of ABA and GA metabolism and signalling in wheat seeds underlies germination response to temperature.