Article Figures & Data
Figures
- FIG. 1.
Minimum-evolution dendrogram of the Haemophilus sensu stricto cluster. The tree, rooted by E. coli strain K-12, is based on concatenated adk, pgi, recA, infB, and 16S rRNA gene sequences, with bootstrap values of ≥50% of 1,000 bootstraps indicated. Node I contains all H. influenzae and hemolytic and nonhemolytic H. haemolyticus strains within an Haemophilus sensu stricto cluster, while nodes II and III contain selected members of the family Pasteurellaceae. Strains with identical sequences are listed on the same branch. Red boxes define beta-hemolytic H. haemolyticus strains. Positive (+) and negative (−) results for the iga gene probe hybridization, P6 OMP MAb 7F3 reactivity, the production of gas during glucose metabolism, and H2S production are shown to the right of each strain. The results are also relative to the strain order on branches with multiple strains. On the basis of the stratification of iga and other results (see the text and Table 2), it is proposed that node A contains all H. influenzae strains, while nodes B to F contain all H. haemolyticus strains.
- FIG. 2.
Multiple and single DNA sequence segregation of H. influenzae and H. haemolyticus strains. Radiation tree views of the (A) five concatenated sequences and individual (B) adk, (C) pgi, (D) recA, (E) infB, and (F) 16S rRNA gene sequences. H. influenzae (purple dots) and H. haemolyticus (red dots) strains are shown together with other members of the family Pasteurellaceae (green triangles).
Tables
- TABLE 1.
Population and ecological stratification of the H. influenzae and H. haemolyticus collections
Epidemiologic segregation (no. of isolates) No. of isolates References H. influenzae (n = 88 total) H. haemolyticus (n = 109 total) Isolation site Middle ear (44) 44 0 12, 29 Healthy throat (107) 44 63 11, 38, 54 Sputum (46) 0 46 40, 50 Age groups Adult (67) 6 61 38, 40, 50 Child (130) 82 48 11, 54 - TABLE 2.
Oligonucleotides used for PCR or DNA sequencing
Gene Primer sequencea Position in Rd genome adk F: GGTGCACCGGGTGCAGGTAA 376521 R: CCTAAGATTTTATCTAACTC 375899 pgi F: GGTGAAAAAATCAATCGTAC 1644657 R: ATTGAAAGACCAATAGCTGA 1645246 recA F: ATGGCAACTCAAGAAGAAAA 622556 R: TTACCAAACATCACGCCTAT 621940 infB F: TGAAAATGAGCTTGAAGAAGCGG 1361550 R: GATAGTTGCCACAGGGCCACGACC 1362198 16S rRNA F1: CCAGCAGCCGCGGTAATACG 624342 R1: ATCGGYTACCTTGTTACGACTTC 625335 F2: GCCCGCACAAGCGGTGGAGCATGTG 624751 R2: CTCGTAAGGGCCATGATGACTTGACG 625039 iga F: GTTCCACCACCTGCGCCTGCTAC 1050386 R: GTTATATTGCCCCTCGTTATTCA 1049130 licAb F: GTAGGATTTGTTAAAACTTGCTACAAGCC 1608693 R: GGCAATTCCTCTAACAGTTTAAATGCTGCG 1609579 lic2Ab F: ATATTACATAATATAGAGGAATCTAG 571382 R: CTACATAAAACGAACAATTTCTTTACC 570690 lgtCb F: CGGACTGTCAGTCAGACAATG 289338 R: CTCAAAATGATCATACCAAGATG 288499 - TABLE 3.
Distribution of taxonomic traits among H. influenzae and H. haemolyticus isolates
Trait No. (%) of isolatesa PRb Result of χ2 analysis (P valued) H. influenzae (n = 88) H. haemolyticus (n = 109) Molecular-based traits iga gene probe hybridization 88 (100) 0 (0.0) Referent MAb 7F3 reactivity 85 (96.6) 13 (11.9) 8.10 <0.0001 Classical traits H2S production 11 (12.5) 75 (68.8) 0.18 <0.0001 Gas production 5 (5.7) 96 (88.1) 0.06 <0.0001 Indole production 73 (83.0) 68 (62.4) 1.33 0.0015 Urease activity 74 (84.1) 77 (70.6) 1.19 0.0265 ODC activity 24 (27.3) 8 (7.3) 3.72 0.0002 Biotype I 18 (20.5) 4 (3.7) 5.57 0.0002 Biotype II 44 (50.0) 43 (39.4) 1.27 0.1382 Biotype III 12 (13.6) 28 (25.7) 0.53 0.0366 Biotype IV 0 (0)c 2 (1.8) 0.31 0.2015 Biotype V 5 (5.7) 2 (1.8) 3.10 0.1470 Biotype VI 1 (1.1) 0 (0)c 2.48 0.2645 Biotype VII 6 (6.8) 19 (17.4) 0.39 0.0261 Biotype VIII 2 (2.3) 11 (10.1) 0.23 0.0280 LOS gene virulence traits licA 84 (95.5) 46 (42.2) 2.26 <0.0001 lic2A 80 (90.0) 17 (15.6) 5.83 <0.0001 lgtC 88 (100.0) 2 (1.8) 54.50 <0.0001 ↵a The species designation is based on the stratification of strains in the MLSA dendrogram of Fig. 1 and on the presence or absence of hybridization with an iga gene probe.
↵b PR, prevalence ratio. The prevalence ratios were calculated for H. influenzae by using H. haemolyticus as the referent group.
↵c Logit, 0.5 was used in place of 0 for prevalence ratio and statistical calculations.
↵d A P value of <0.05 is considered statistically significant.
- TABLE 4.
Trends of species-specific traits within a species
Trait No. (%) of strainsa H. influenzae nodes H. haemolyticus nodes A1 (n = 72) A2 and A3 (n = 16) B to E (n = 14) F (n = 95) H2S production 4 (5.6) 7 (43.8)c 7 (50.0) 68 (71.6) Indole production 63 (87.5) 10 (62.5)c 10 (71.4) 58 (61.1) licA 72 (100) 12 (75.0)c 8 (57.1) 38 (40.0) lic2A 71 (98.6) 9 (56.3)c 3 (21.4) 14 (14.7) P6 positive 70 (97.2) 15 (93.7) 3 (21.4) 10 (10.5) lgtC 72 (100) 16 (100) 0 (0)b 2 (2.1) Gas production 4 (5.6) 1 (6.3) 12 (85.7) 84 (88.4) Urease activity 59 (81.9) 15 (93.8) 6 (42.8) 71 (74.7)c ODC activity 16 (22.2) 8 (50.0)c 1 (7.1) 7 (7.4) - TABLE 5.
Distribution of taxonomic traits between hemolytic and nonhemolytic H. haemolyticus strains
Trait No. (%) of strains PRa Results of χ2 analysis (P valuec) Hemolytic (n = 33) Nonhemolytic (n = 76) Molecular-based traits Hemolysis 33 (100) 0 (0) Referent MAb 7F3 0 (0)b 13 (17.1) 11.3 0.0114 Traditional traits H2S production 24 (72.7) 51 (67.1) 0.92 0.5605 Gas production 31 (93.9) 65 (85.5) 0.88 0.2131 Indole production 20 (60.6) 48 (63.2) 1.04 0.8005 Urease activity 28 (84.8) 49 (64.5) 0.76 0.0319 ODC activity 2 (6.1) 6 (7.9) 1.30 0.7358 LOS gene virulence traits licA 8 (24.2) 38 (50.0) 2.06 0.0124 lic2A 1 (3.0) 16 (21.1) 6.95 0.0172 lgtC 0 (0)b 2 (2.6) 1.74 0.3469
