Patients with Chronic Obstructive Pulmonary Disease harbour a variation of Haemophilus species

Abstract

H. haemolyticus is often misidentified as NTHi due to their close phylogenetic relationship. Differentiating between the two is important for correct identification and appropriate treatment of infective organism and to ensure any role of H. haemolyticus in disease is not being overlooked. Speciation however is not completely reliable by culture and PCR methods due to the loss of haemolysis by H. haemolyticus and the heterogeneity of NTHi. Haemophilus isolates from COPD as part of the AERIS study (ClinicalTrials - NCT01360398) were speciated by analysing sequence data for the presence of molecular markers. Further investigation into the genomic relationship was carried out using average nucleotide identity and phylogeny of allelic and genome alignments. Only 6.3% were identified as H. haemolyticus. Multiple in silico methods were able to distinguish H. haemolyticus from NTHi. However, no single gene target was found to be 100% accurate. A group of omp2 negative NTHi were observed to be phylogenetically divergent from H. haemolyticus and remaining NTHi. The presence of an atypical group from a geographically and disease limited set of isolates supports the theory that the heterogeneity of NTHi may provide a genetic continuum between NTHi and H. haemolyticus.

Figure 1

Allelic variation of (A) smpB, (B) omp6 and (C) hpd from H. influenzae and H. haemolyticus. Unrooted maximum – likelihood phylogenetic trees constructed in RaxML and visualised in microreact. MUSCLE alignments of sequences for smpB, omp6 and hpd from 1460 Haemophilus spp. where present using the GTRGAMMA model for nucleotide substitution. Scale bar indicates number of nucleotide substitutions per site. Colours indicate study classification group as per legend. Reference NCTC 10839 and NCTC 10659 are shown in green for H. haemolyticus and cluster within the H. haemolyticus clade. NTHi reference isolate NCTC 4842 clusters within an NTHi clade in hpd as marked. URL to access phylogeny and metadata available from Supplementary Table 2.

Figure 2

MetaPhlAn results for 1460 Haemophilus spp isolated from 24 COPD patients from 134 visits. Genetic similarity is based on percentage similarity of the Haemophilus isolates to 260 reference genes for H. influenzae. NTHi and f- NTHi can be seen to share a high degree of identity compared to Group III and H. haemolyticus.

Figure 3

Heatmap summarising genetic similarity ANI calculations between 388 representative samples of Haemophilus spp isolated from 24 COPD patients over two years. Blue indicates a similarity of less than 94% and therefore a distinct species. Here this represents the H. haemolyticus isolates as compared to NTHi. Dark red areas depict isolates of high genetic similarity and the dendrograms show a clustering based on ST. The lighter area represents the Group III NTHi.

Figure 4

Maximum-likelihood phylogeny from core genome alignments of representative subset of 388 Haemophilus spp isolated from 24 COPD patients during the AERIS Study. Group III are distant from the majority of the NTHi however in this instance the Group III clade also contains NTHi STs 311, 704 and 513. The fucK negative isolates are observed to cluster with the majority of the NTHi and H. haemolyticus cluster together but distinctly from NTHi. Bar represents number of nucleotide substitutions per site. URL to access phylogeny and metadata available from Supplementary Table 2.