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. 1999 Jul;37(7):2142-7.
doi: 10.1128/JCM.37.7.2142-2147.1999.

Subtyping of Haemophilus influenzae strains by pulsed-field gel electrophoresis

M Saito  1 , A Umeda, S i Yoshida
Affiliations

Subtyping of Haemophilus influenzae strains by pulsed-field gel electrophoresis

M Saito et al. J Clin Microbiol. 1999 Jul.

Abstract

A total of 200 isolates of Haemophilus influenzae were analyzed by serotyping, biotyping, and pulsed-field gel electrophoresis (PFGE). A total of 178 epidemiologically unrelated strains of H. influenzae demonstrated a variety of genome patterns by PFGE, and 165 genotypes were thus obtained in this study. PFGE typing proved to have a much stronger discriminatory power than either serotyping or biotyping. Six serotype b strains were all classified into discrete genotypes. A PFGE analysis of 18 strains obtained from the nasopharynx, blood, and cerebrospinal fluid of patients with meningitis also supported the hypothesis that invasive H. influenzae disseminates from the nasopharynx to the bloodstream and then subsequently to other body sites. PFGE typing of 10 other strains isolated from household contacts of patients with H. influenzae infection revealed that the strain that caused the H. influenzae infection often colonized the nasopharynges of household contacts. Our findings suggest that PFGE analysis is useful for the epidemiological study of H. influenzae infection, even when the invasive disease is caused by serotype b strains.

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Figures

FIG. 1
FIG. 1
Examples of PFGE patterns of chromosomal DNAs extracted from clinical H. influenzae isolates. Chromosomal DNA was digested with the SmaI restriction endonuclease. Lanes M, bacteriophage lambda concatamer molecular size marker. (A) Patterns of 20 strains of biotype I. Nineteen different patterns were observed, and a pair with the same pattern (lanes 5 and 7) was found. Lanes: 1 and 2, serotype a; 3 to 6, serotype b; 7, serotype c; 8 to 20, nontypeable (NT) strains (lanes 8 to 14, strains isolated from the nasopharynx; lane 15, strain isolated from sputum; lane 16, strain isolated from eye mucus; lanes 17 and 18, strains isolated from middle ear mucus; lanes 19 and 20, strains isolated from urine). (B) Patterns of 20 strains of biotype II. The strains in lanes 7 and 13 showed the same genome patterns, and the strains in lanes 9, 10, and 12 and in lanes 11 and 15 also showed the same genome patterns. Therefore, 16 different patterns are shown in panel B. Lanes: 1 and 2, serotype b; 3 to 20, nontypeable strains (lanes 3 to 10, strains isolated from the nasopharynx; lanes 11 to 14, strains isolated from sputum; lane 15, strain isolated from eye mucus; lane 16, strain isolated from middle-ear mucus; lanes 17 and 18, strain isolated from urine; lane 19, strain isolated from vaginal mucus; lane 20, strain isolated from CSF). (C) Patterns of 20 strains of biotype III. The strains in lanes 17 and 20 had the same genome patterns; therefore, 19 different patterns are shown in panel C. All strains were nontypeable by serotyping. Lanes: 1 to 11, strains isolated from nasopharynx; lanes 12 to 15, strains isolated from sputum; lane 16, strain isolated from eye mucus; 17 to 19, strains isolated from middle-ear mucus; 20, strain isolated from urine. (D) Patterns of 19 strains of biotypes IV to VIII. All strains except the one in lane 5 (serotype A) were nontypeable by serotyping. Lanes: 1 to 4, biotype IV strains isolated from the nasopharynx; 5 to 9, biotype V strains (lanes 5 and 6, strains isolated from the nasopharynx; lanes 7 and 8, strains isolated from sputum; 9, strains isolated from middle-ear mucus); 10, biotype VI strain isolated from nasopharynx; 11 to 18, biotype VII strains isolated from nasopharynx; 19, biotype VIII strain isolated from sputum.
FIG. 1
FIG. 1
Examples of PFGE patterns of chromosomal DNAs extracted from clinical H. influenzae isolates. Chromosomal DNA was digested with the SmaI restriction endonuclease. Lanes M, bacteriophage lambda concatamer molecular size marker. (A) Patterns of 20 strains of biotype I. Nineteen different patterns were observed, and a pair with the same pattern (lanes 5 and 7) was found. Lanes: 1 and 2, serotype a; 3 to 6, serotype b; 7, serotype c; 8 to 20, nontypeable (NT) strains (lanes 8 to 14, strains isolated from the nasopharynx; lane 15, strain isolated from sputum; lane 16, strain isolated from eye mucus; lanes 17 and 18, strains isolated from middle ear mucus; lanes 19 and 20, strains isolated from urine). (B) Patterns of 20 strains of biotype II. The strains in lanes 7 and 13 showed the same genome patterns, and the strains in lanes 9, 10, and 12 and in lanes 11 and 15 also showed the same genome patterns. Therefore, 16 different patterns are shown in panel B. Lanes: 1 and 2, serotype b; 3 to 20, nontypeable strains (lanes 3 to 10, strains isolated from the nasopharynx; lanes 11 to 14, strains isolated from sputum; lane 15, strain isolated from eye mucus; lane 16, strain isolated from middle-ear mucus; lanes 17 and 18, strain isolated from urine; lane 19, strain isolated from vaginal mucus; lane 20, strain isolated from CSF). (C) Patterns of 20 strains of biotype III. The strains in lanes 17 and 20 had the same genome patterns; therefore, 19 different patterns are shown in panel C. All strains were nontypeable by serotyping. Lanes: 1 to 11, strains isolated from nasopharynx; lanes 12 to 15, strains isolated from sputum; lane 16, strain isolated from eye mucus; 17 to 19, strains isolated from middle-ear mucus; 20, strain isolated from urine. (D) Patterns of 19 strains of biotypes IV to VIII. All strains except the one in lane 5 (serotype A) were nontypeable by serotyping. Lanes: 1 to 4, biotype IV strains isolated from the nasopharynx; 5 to 9, biotype V strains (lanes 5 and 6, strains isolated from the nasopharynx; lanes 7 and 8, strains isolated from sputum; 9, strains isolated from middle-ear mucus); 10, biotype VI strain isolated from nasopharynx; 11 to 18, biotype VII strains isolated from nasopharynx; 19, biotype VIII strain isolated from sputum.
FIG. 2
FIG. 2
PFGE patterns of isolates from six patients (patients 1 to 6) with meningitis. The strains were isolated from CSF (lanes C), pharynx (lanes P), nasal cavity (lanes N), and blood (lanes B). Lane M, molecular size marker. Patients 1 and 2 had meningitis caused by serotype b, biotype I strains, patients 3 and 4 had meningitis caused by serotype b, biotype II strains, patient 5 had meningitis caused by a serotype c, biotype I strain, and patient 6 had meningitis caused by a nonencapsulated biotype II strain.
FIG. 3
FIG. 3
PFGE patterns of isolates from seven patients with H. influenzae infection (∗) and their household contacts (no mark). Patients 1, 3, and 11 had meningitis; patients 7, 8, 9, and 10 had respiratory tract infections. Patients 1 and 3 are the same as patients 1 and 3 in Fig. 2.
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