Reference system for characterization of Bordetella pertussis pulsed-field gel electrophoresis profiles

Abstract

Pulsed-field gel electrophoresis (PFGE) has been used as an epidemiological tool for surveillance studies of Bordetella pertussis since the early 1990s. To date there is no standardized procedure for comparison of results, and therefore it has been difficult to directly compare PFGE results between laboratories. We propose a profile-based reference system for PFGE characterization of B. pertussis strain variation and to establish traceability of B. pertussis PFGE results. We initially suggest 35 Swedish reference strains as reference material for PFGE traceability. This reference material is deposited at the Culture Collection of the University of Gothenburg, Gothenburg, Sweden. Altogether, 1,810 Swedish clinical isolates from between 1970 and 2003 were studied, together with the Swedish Pw vaccine strain, six reference strains, and two U.S. isolates. Our system provides evidence that profiles obtained by using only one enzyme, i.e., XbaI, give enough data to analyze the epidemiological relationship between them. Characterization with one enzyme is far less labor intensive, yielding results in half the time than when a two-enzyme procedure is used. Also, we can see that there is a correlation between PFGE profile and pertactin type. One common PFGE profile, BpSR11 (n = 455), showed 100% prn2 and 100% Fim3 when analyzed for pertactin type and serotype. On the other hand, strains with the same profile may express various serotypes when isolated over longer periods of time. Subculturing of the same isolate eight times or lyophilization caused no change in PFGE profile.

FIG. 1.

PFGE gel. Lane 1, Swedish vaccine strain. Lanes 3, 10, and 18, low-range marker. Lane 11, strain T41, which was applied in every gel as an intragel quality control. All other lanes show clinical test strains. High band resolution can be seen in the area also between 48.5 and 97.5 kb.

FIG. 2.

PFGE gel. From left to right, profiles of BpSR16, BpSR18, and the low-range PFGE DNA marker (New England BioLabs). The only band difference between the two profiles is near the 97.0-kb region indicated by the white arrow.

FIG. 3.

Classification of the 35 prevalent Swedish profiles, including the six reference strains (indicated by asterisks). UPGMA with 1% band tolerance and 1% optimization settings was used as the clustering method. Only DNA profiles obtained after restriction with XbaI are shown. The serotype (Fim) and pertactin type (PRN) represent the most prevalent types within the profile. Gp, grouping system described by Weber et al. (29).

FIG. 4.

Isolates with the BpSR18 profile express different fimbria serotypes during periods with different vaccination schedules.

FIG. 5.

Strain 44122 was used for production of the Swedish whole-cell vaccine from the 1950s to 1978. PFGE of several clones from 11 different vials containing control material for vaccine production during the 8-year period from 1956 to 1964 showed heterogeneity of profiles. At least four different profiles could be documented (the profiles are identified by suffixes indicating year/month).

FIG. 6.

Dendrogram showing six strains (C1 to C6) subcultured for eight passages (P1 to P8), with Cn and Pn indicating the strain and passage number, respectively. No change in the pulsed-field profile could be identified. UPGMA with 1% band tolerance and 1% optimization settings was used as the clustering method. Only DNA profiles obtained after restriction with XbaI are shown.