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Bacteriology

Relationships of Nontypeable Haemophilus influenzae Strains to Hemolytic and Nonhemolytic Haemophilus haemolyticus Strains

Kirk W. McCrea, Jingping Xie, Nathan LaCross, Mayurika Patel, Deepa Mukundan, Timothy F. Murphy, Carl F. Marrs, Janet R. Gilsdorf
Kirk W. McCrea
1Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan
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  • For correspondence: sigmb@med.umich.edu
Jingping Xie
1Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan
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Nathan LaCross
1Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan
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Mayurika Patel
1Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan
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Deepa Mukundan
1Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan
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Timothy F. Murphy
3Department of Medicine, University at Buffalo, State University of New York, Buffalo, New York
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Carl F. Marrs
2Department of Epidemiology, University of Michigan School of Public Health, Ann Arbor, Michigan
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Janet R. Gilsdorf
1Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan
2Department of Epidemiology, University of Michigan School of Public Health, Ann Arbor, Michigan
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DOI: 10.1128/JCM.01832-07
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  • FIG. 1.
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    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.
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    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

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  • TABLE 1.

    Population and ecological stratification of the H. influenzae and H. haemolyticus collections

    Epidemiologic segregation (no. of isolates)No. of isolatesReferences
    H. influenzae (n = 88 total)H. haemolyticus (n = 109 total)
    Isolation site
        Middle ear (44)440 12, 29
        Healthy throat (107)4463 11, 38, 54
        Sputum (46)046 40, 50
    Age groups
        Adult (67)661 38, 40, 50
        Child (130)8248 11, 54
  • TABLE 2.

    Oligonucleotides used for PCR or DNA sequencing

    GenePrimer sequenceaPosition in Rd genome
    adk F: GGTGCACCGGGTGCAGGTAA376521
    R: CCTAAGATTTTATCTAACTC375899
    pgi F: GGTGAAAAAATCAATCGTAC1644657
    R: ATTGAAAGACCAATAGCTGA1645246
    recA F: ATGGCAACTCAAGAAGAAAA622556
    R: TTACCAAACATCACGCCTAT621940
    infB F: TGAAAATGAGCTTGAAGAAGCGG1361550
    R: GATAGTTGCCACAGGGCCACGACC1362198
    16S rRNAF1: CCAGCAGCCGCGGTAATACG624342
    R1: ATCGGYTACCTTGTTACGACTTC625335
    F2: GCCCGCACAAGCGGTGGAGCATGTG624751
    R2: CTCGTAAGGGCCATGATGACTTGACG625039
    iga F: GTTCCACCACCTGCGCCTGCTAC1050386
    R: GTTATATTGCCCCTCGTTATTCA1049130
    licA b F: GTAGGATTTGTTAAAACTTGCTACAAGCC1608693
    R: GGCAATTCCTCTAACAGTTTAAATGCTGCG1609579
    lic2A b F: ATATTACATAATATAGAGGAATCTAG571382
    R: CTACATAAAACGAACAATTTCTTTACC570690
    lgtC b F: CGGACTGTCAGTCAGACAATG289338
    R: CTCAAAATGATCATACCAAGATG288499
    • ↵ a All oligonucleotides are based on the DNA sequences from H. influenzae strain Rd. F, forward, R, reverse.

    • ↵ b The forward primers begin downstream of tetranucleotide repeats.

  • TABLE 3.

    Distribution of taxonomic traits among H. influenzae and H. haemolyticus isolates

    TraitNo. (%) of isolatesaPRbResult of χ2 analysis (P valued)
    H. influenzae (n = 88)H. haemolyticus (n = 109)
    Molecular-based traits
        iga gene probe hybridization88 (100)0 (0.0)Referent
        MAb 7F3 reactivity85 (96.6)13 (11.9)8.10<0.0001
    Classical traits
        H2S production11 (12.5)75 (68.8)0.18<0.0001
        Gas production5 (5.7)96 (88.1)0.06<0.0001
        Indole production73 (83.0)68 (62.4)1.330.0015
        Urease activity74 (84.1)77 (70.6)1.190.0265
        ODC activity24 (27.3)8 (7.3)3.720.0002
        Biotype I18 (20.5)4 (3.7)5.570.0002
        Biotype II44 (50.0)43 (39.4)1.270.1382
        Biotype III12 (13.6)28 (25.7)0.530.0366
        Biotype IV0 (0)c2 (1.8)0.310.2015
        Biotype V5 (5.7)2 (1.8)3.100.1470
        Biotype VI1 (1.1)0 (0)c2.480.2645
        Biotype VII6 (6.8)19 (17.4)0.390.0261
        Biotype VIII2 (2.3)11 (10.1)0.230.0280
    LOS gene virulence traits
        licA84 (95.5)46 (42.2)2.26<0.0001
        lic2A80 (90.0)17 (15.6)5.83<0.0001
        lgtC88 (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

    TraitNo. (%) of strainsa
    H. influenzae nodesH. haemolyticus nodes
    A1 (n = 72)A2 and A3 (n = 16)B to E (n = 14)F (n = 95)
    H2S production4 (5.6)7 (43.8)c7 (50.0)68 (71.6)
    Indole production63 (87.5)10 (62.5)c10 (71.4)58 (61.1)
    licA 72 (100)12 (75.0)c8 (57.1)38 (40.0)
    lic2A 71 (98.6)9 (56.3)c3 (21.4)14 (14.7)
    P6 positive70 (97.2)15 (93.7)3 (21.4)10 (10.5)
    lgtC 72 (100)16 (100)0 (0)b2 (2.1)
    Gas production4 (5.6)1 (6.3)12 (85.7)84 (88.4)
    Urease activity59 (81.9)15 (93.8)6 (42.8)71 (74.7)c
    ODC activity16 (22.2)8 (50.0)c1 (7.1)7 (7.4)
    • ↵ a The nodes and the number of strains in each node are relative to those in the dendrogram in Fig. 1.

    • ↵ b Logit, 0.5 was used instead of 0 for statistical calculations.

    • ↵ c Nodes possessing a statistically significant difference (P < 0.05) for the trait on the basis of Student's t test.

  • TABLE 5.

    Distribution of taxonomic traits between hemolytic and nonhemolytic H. haemolyticus strains

    TraitNo. (%) of strainsPRaResults of χ2 analysis (P valuec)
    Hemolytic (n = 33)Nonhemolytic (n = 76)
    Molecular-based traits
        Hemolysis33 (100)0 (0)Referent
        MAb 7F30 (0)b13 (17.1)11.30.0114
    Traditional traits
        H2S production24 (72.7)51 (67.1)0.920.5605
        Gas production31 (93.9)65 (85.5)0.880.2131
        Indole production20 (60.6)48 (63.2)1.040.8005
        Urease activity28 (84.8)49 (64.5)0.760.0319
        ODC activity2 (6.1)6 (7.9)1.300.7358
    LOS gene virulence traits
        licA8 (24.2)38 (50.0)2.060.0124
        lic2A1 (3.0)16 (21.1)6.950.0172
        lgtC0 (0)b2 (2.6)1.740.3469
    • ↵ a PR, prevalence ratio. Prevalence ratios were calculated for nonhemolytic strains by using the hemolytic strains as the referent group.

    • ↵ b Logit, 0.5 was used instead of 0 for prevalence ratio and statistical calculations.

    • ↵ c A P value of <0.05 is considered statistically significant.

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Relationships of Nontypeable Haemophilus influenzae Strains to Hemolytic and Nonhemolytic Haemophilus haemolyticus Strains
Kirk W. McCrea, Jingping Xie, Nathan LaCross, Mayurika Patel, Deepa Mukundan, Timothy F. Murphy, Carl F. Marrs, Janet R. Gilsdorf
Journal of Clinical Microbiology Feb 2008, 46 (2) 406-416; DOI: 10.1128/JCM.01832-07

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Relationships of Nontypeable Haemophilus influenzae Strains to Hemolytic and Nonhemolytic Haemophilus haemolyticus Strains
Kirk W. McCrea, Jingping Xie, Nathan LaCross, Mayurika Patel, Deepa Mukundan, Timothy F. Murphy, Carl F. Marrs, Janet R. Gilsdorf
Journal of Clinical Microbiology Feb 2008, 46 (2) 406-416; DOI: 10.1128/JCM.01832-07
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KEYWORDS

Bacterial Proteins
Haemophilus

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