Characterization of Non-Type B Haemophilus influenzae Strains Isolated from Patients with Invasive Disease

Bacterial strains.Forty-one non-Hib isolates recovered from patients with invasive disease in Italy, between April 1994 and December 1998, were analyzed. Thirty-two strains were obtained from cases detected through the Active Surveillance Program on H. influenzae Invasive Disease; 9 other strains were isolated from cases reported to the National Surveillance System for Bacterial Meningitis (4). One Hib isolate belonging to the clone endemically present in Italy (27) was also included in the study.

Capsular genotyping of H. influenzae isolates and characteristics of cases.For PCR capsular genotyping, two separate amplifications of the target DNA were carried out. In a first round of PCR, primers omp1 and omp2 (11) directed to theompP2 gene were used to confirm the H. influenzaespecies, while primers HI-1 and HI-2 (7) directed to thebexA region proved capsulation. In a second round of PCR, primers directed to each capsule type-specific region (7) were used. Primers were supplied by M-Medical, Florence, Italy. Preparations of total DNA and amplification reactions were performed as previously described (7). Samples underwent 25 cycles in a Perkin-Elmer Cetus 9600 instrument with the following parameters: denaturation at 94°C (1 min), annealing at 55°C (1 min), elongation at 72°C (1 min), and finally 8 min of incubation at 72°C. The resulting PCR products were electrophoresed through 1.5% agarose (Roche Diagnostics GmbH, Mannheim, Germany) in Tris-borate-EDTA buffer and visualized by ethidium bromide staining. By this method, 33 nonencapsulated strains, 4 type f isolates, and 4 b⁻strains were identified among our isolates. The 33 cases due to nonencapsulated strains were equally distributed among sexes. The median age was 52 years (range, 5 months to 91 years). Bacteremia with other nonidentified foci was the most frequent clinical presentation (41.4%), followed by meningitis (31%). Of the cases, 31% presented underlying pathologies, such as cancer. The case fatality rate was 14.3%. All but one of the invasive diseases due to H. influenzae type f strains occurred in adult males. On the other hand, three out of the four invasive diseases caused by b⁻strains were observed in children under 6 years of age. Meningitis was the most frequent diagnosis in both type f and b⁻ invasive infections.

PFGE.PFGE was performed by following the procedures previously described (27). All isolates were analyzed using the restriction enzyme SmaI (Roche Diagnostics GmbH). Representative PFGE patterns obtained for some nonencapsulated strains are shown in Fig. 1. Thirty different patterns were found among the 33 nonencapsulated isolates. Twenty-seven (90%) were found for single isolates, and each of the other three (10%) was found for two isolates. According to the interpreting criteria reported by Tenover et al. (28), 19 strains showing an individual pattern were considered totally unrelated to each other, whereas 8 showed some degree of relatedness. In particular, six strains isolated from patients from different towns in Lombardia, between October 1997 and January 1998, represented a group of genetically related isolates (data not shown). All of the nonencapsulated strains studied were unrelated to the invasive Hib strain circulating in Italy (Fig. 1). A close genetic relationship was observed among the fourH. influenzae type f strains. Two of them shared the same restriction pattern (hereafter termed pattern A), while the other two showed profiles which can be considered, respectively, closely and possibly related to pattern A (Fig. 2). Likewise (and hereafter designated pattern B), a similar close genetic relationship was found among the four b⁻ strains (Fig. 2). The H. influenzae type f strains tested can be considered unrelated to the Hib strain, while the b⁻ strains analyzed were always related to Hib.

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Fig. 1.

Examples of PFGE patterns of chromosomal DNAs extracted from nonencapsulated H. influenzae isolates. Chromosomal DNA was digested with the SmaI restriction endonuclease. Lanes: 1, Hib strain belonging to the clone endemically present in Italy; 2 through 10, nonencapsulated H. influenzae strains; M, lamba ladder pulsed-field gel marker. This figure shows nine different patterns among nonencapsulated H. influenzae isolates; all of these isolates were unrelated to the Hib strain.

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Fig. 2.

PFGE patterns of SmaI-digested chromosomal DNAs of H. influenzae isolates. Capsular type designations are indicated above lanes. M, lamba ladder pulsed-field gel marker. The strains in lanes 4 and 5 showed indistinguishable profiles (pattern B); the strains in lanes 2 and 3 were, respectively, possibly and closely related to pattern B. The strains in lanes 6 and 8 had the same pattern (pattern A), whereas the strains in lanes 7 and 9 were, respectively, closely or possibly related to pattern A.

Fimbrial expression and detection of the hifA gene by PCR.Detection of hemagglutinating fimbriae was carried out by a semiquantitative assay for the ability to agglutinate human erythrocytes as described elsewhere (19). The specificity of hemagglutination for fimbriae was assessed by inhibition with the sialylated ganglioside GM1 (Sigma, St. Louis, Mo.) at a final concentration of 100 μg/ml (31). Five nonencapsulated isolates were positive in the hemagglutination assay, showing titers ranging from 1:8 to 1:16. The presence of the hifA gene, which encodes the major subunit of the hemagglutinating fimbriae ofH. influenzae, was determined by PCR, using primers fgHifA1 and fgHifA2 as described by F. Geluk et al. (9). Primers were supplied by M-Medical. Amplification reactions were performed under the conditions previously described (9). Hib strains 770235 and 760705 (kindly provided by P. van Ulsen, RIVM, Bilthoven, The Netherlands) were used as positive and negative controls, respectively. The presence of the hifA gene was revealed by the generation of an 800-bp product in all five of the hemagglutinating isolates and in another seven strains which did not express fimbriae (Fig. 3). Those hemagglutinating negative isolates containing an hifA gene included one nonencapsulated, two b⁻, and all four type f strains.

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Fig. 3.

Agarose gel electrophoresis of PCR products fromH. influenzae strains amplified with fgHifA1 and fgHifA2 primers. Lanes: 1 to 6, nonencapsulated H. influenzaestrains; 7 to 10, H. influenzae type f strains; 11 and 12, b⁻ strains; 13, Hib 770235, a positive control; M, DNA molecular mass marker. The generation of an 800-bp product indicated the presence of the hifA gene. This figure shows thehifA-positive strains recovered in this study.

Ampicillin susceptibility testing.The minimum inhibitory concentrations (MICs) for ampicillin were determined by E-test (AB Biodisk, Solna, Sweden) using Haemophilus Test Medium agar plates incubated at 37°C for 20 h in a humid atmosphere enriched with 5% CO₂. Reference Hib strain ATCC 10211 was used as the control. The interpretative breakpoints used were based on NCCLS criteria (18). Production of β-lactamase was detected by the cefinase disk test (Becton-Dickinson, Cockeysville, Md.). The MIC at which 90% of the isolates tested wre inhibited (MIC₉₀) for the 33 nonencapsulated strains was 0.5 μg/ml (range, 0.25 to 8 μg/ml). Thirty-one nonencapsulated strains were susceptible, one was intermediately resistant (MIC, 2 μg/ml), and one was resistant (MIC, 8 μg/ml) to ampicillin. The last strain, isolated in 1998, corresponded to 5% of the nonencapsulated strains isolated that year. All four of the H. influenzae type f isolates and the four b⁻ strains were found to be susceptible to the same MIC, 0.25 μg/ml. No strains were found that produced β-lactamase.

The 41 non-type b H. influenzae isolates analyzed in this study were first tested by a PCR genotyping method for the unequivocal assignment of their capsular type. The results obtained indicated that nonencapsulated isolates account for most of the non-type b strains that cause invasive disease in Italy. Moreover, the recovery of b⁻ strains confirmed the usefulness of PCR capsular genotyping (3). An analysis of cases revealed that invasive disease caused by nonencapsulated and type f strains occurred mostly in adults with diagnosed bacteremia, thus differing from the pattern produced by Hib isolates. On the contrary, the presentation of b⁻ disease may resemble that of Hib, although the number of b⁻ strains recovered in the study is too small to be conclusive.

To study the genetic relationships among the different H. influenzae isolates, we used PFGE, a well-established methodology, which already has been used to differentiate H. influenzaestrains (15, 22, 27). The invasive nonencapsulated isolates that we analyzed showed considerable genetic heterogeneity, confirming the earlier described genetic diversity of nonencapsulated H. influenzae strains (16, 26, 30). This result extends the data obtained by van Alphen et al. (30) to invasive disease; this study revealed no significant association between specific multilocus genotypes and kinds of disease.

A close genetic relationship was found both among the H. influenzae type f strains and among the b⁻ strains recovered, although the isolates analyzed were few. As expected, the b⁻ strains tested were genetically related to the invasive Hib strain circulating in Italy. Conversely, the four type f strains were totally unrelated to the same Hib isolate. This finding is in accordance with the data reported by Musser et al. (17) that type c, e, and f strains have no close genetic relationships to strains of other serotypes.

Hemagglutinating fimbriae can be present on both encapsulated and nonencapsulated H. influenzae strains, and they have been shown to mediate adherence to human erythrocytes carrying the AnWj antigen and to specific human epithelial cells by binding to the GM1-like receptor (10, 21). A fimbria gene cluster containing the genes hifA to hifE has been identified (14, 33). It has been reported that the expression of hemagglutinating fimbriae is subject to reversible phase variation (32); during natural infection with Hib, nasopharyngeal isolates are often fimbriated while their isogenic counterparts from blood or cerebrospinal fluid are invariably found to be nonfimbriated (19). Our results seem to indicate that type f and b⁻ strains behave as Hib strains; that is, systemic isolates do not express fimbriae even if fimbria genes are present. As far as nonencapsulated H. influenzae strains are concerned and contrary to results from other studies regarding strains isolated from patients with noninvasive diseases (9, 13), our data demonstrated that nonencapsulated strains isolated from systemic sites generally express fimbriae, if a fimbria gene cluster is present. No association between expression of fimbriae and clinical presentation of the disease was observed. Whether a correlation between the expression of fimbriae and the invasive properties of a subset of nonencapsulated strains may be supposed is still unclear, and much additional work will be required in this area.

In the last decade, H. influenzae strains resistant to ampicillin have been isolated with increasing frequency worldwide (5, 8). Plasmid-mediated production of TEM or ROB β-lactamases is the most common mechanism of resistance to ampicillin; however, resistance can also be caused by intrinsic mechanisms involving target modification. Although strains that are β-lactamase negative and ampicillin resistant (BLNAR) are relatively uncommon, their numbers are increasing, mainly among nonencapsulated strains (2, 6, 24, 36).

In the present study, no strains were β-lactamase producers. This result is not surprising since β-lactamase production is more frequent among Hib strains (20), and even there, very few β-lactamase-positive strains have been previously reported in Italy (27). Only one nonencapsulated strain fulfilled the definition of BLNAR; nevertheless this result shows that BLNAR strains may be recovered among invasive nonencapsulatedH. influenzae strains in Italy. Little is known regarding the clinical relevance of the BLNAR strains; however, our data suggest the need to monitor both the β-lactamase- and non-β-lactamase-mediated resistance mechanisms in nonencapsulatedH. influenzae isolates.