Supplementary Materials For "Genomic diversity and antimicrobial resistance of Haemophilus colonising the airways of young children with cystic fibrosis"

Abstract. Respiratory infection during childhood is a key risk factor in early cystic fibrosis (CF) lung disease progression. Haemophilus influenzae (Hi) and Haemophilus parainfluenzae (Hpi) are routinely isolated from the lungs of children with CF, however little is known about the frequency and characteristics of Haemophilus colonisation in this context. Here, we describe detection, antimicrobial resistance (AMR), and genome sequencing of Hi/Hpi isolated from airway sampling of 147 participants aged ≤12 years enrolled in the AREST CF program, Melbourne, Australia. The frequency of colonisation per visit was 4.6% for Hi and 32.1% for Hpi, 80.3% of participants had Hi and/or Hpi detected on at least one visit, and using genomic data we estimate 15.6% of participants had persistent colonisation with the same strain for at least two consecutive visits. Isolates were genetically diverse and AMR was common, with 52% of Hi and 82% of Hpi displaying resistance to at least one drug. The genetic basis for AMR could be identified in most cases; putative novel determinants include a new plasmid encoding bla_TEM-1 (ampicillin resistance), a new inhibitor-resistant bla_TEM allele (augmentin resistance), and previously unreported mutations in chromosomally-encoded genes (pbp3, ampicillin resistance; folA/folP, co-trimoxazole resistance; rpoB, rifampicin resistance). Acquired AMR genes were more common in Hpi than Hi (51% vs 21%, p=0.0107) and were mostly associated with the ICEHin mobile element carrying bla_TEM-1, resulting in more ampicillin resistance in Hpi (73% vs 30%, p=0.0004). Genomic data identified six potential instances of Haemophilus transmission between participants, three of which involved participants who shared clinic visit days.

Importance. Cystic fibrosis (CF) lung disease begins during infancy and acute respiratory infections increase the risk of early disease development and progression. Microbes involved in advanced stages of CF are well characterised but less is known about early respiratory colonisers. We report the population dynamics and genomic determinants of AMR in two early coloniser species, Haemophilus influenzae (Hi) and Haemophilus parainfluenzae (Hpi), collected from a paediatric CF cohort. This investigation also reveals Hpi to have a high frequency of AMR encoded on mobile elements that may act as a potential reservoir for emergence and spread of AMR to Hi, which has greater clinical significance as a respiratory pathogen in children. This study provides insight into the evolution of AMR and the colonisation of Hi and Hpi in a paediatric CF cohort, which will help inform future treatment.