Culture-independent Multilocus Sequence Typing (MLST) screening for Haemophilus influenzae cross-infection in non-cystic fibrosis bronchiectasis (NCFB)

There is some evidence of Pseudomonas aeruginos across-infection between patients with non-cystic fibrosis bronchiectasis (NCFB), and clear evidence in Cystic Fibrosis. Haemophilus influenzae (H. influenzae) is the more common pathogen in NFCB patients, yet cross-infectionremains unexplored. We present the novel application of culture-independent Multilocus Sequence Typing (MLST) to screen for cross-infection of H. influenzae in NCFB in both culture-positive and -negative samples.

Methods

We interrogated DNA from 32 sputum samples (26 patients) in our NCFB biorepository, who were known to have H.influenzae in their sputum by preceding 16S rRNA sequencing. Fragments of 7 H. influenzae housekeeping genes were amplified and sequenced. Sequence types were allocated via the MLST scheme. For 5 patients, multiple sputum samples taken at least 4 months apart were assessed longitudinally.

Culture-independent MLST identified 31 of 32 sputum samples as harboring H. influenzae. Of these, 26 were positive for H. influenzae using culture methods. 25 of the 26 culture-positive samples were MLST positive. All 6 culture-negative samples were MLST positive. A MLST sequence type (ST) was allocated to 27 of 32 sputum samples. Five patients had multiple sputum samples with matching STs, indicating strain stability and the consistency of MLST. Two patients who were known household contacts had matching STs and possibly transmitted H.influenzae in their household. The remaining 15 STs were unique, suggesting no evidence of cross-infection.

Culture-independent MLST identifies H. influenzae in culture-negative patients with NCFB and is a potential screening tool for cross-infection. This study did not reveal potential cross-infection events in this cohort.