Bordetella pertussis Clones Identified by Multilocus Variable-Number Tandem-Repeat Analysis

Multilocus variable-number tandem-repeat analysis (MLVA) of 316 Bordetella pertussis isolates collected over 40 years from Australia and 3 other continents identified 66 MLVA types (MTs), including 6 predominant MTs. Typing of genes encoding acellular vaccine antigens showed changes that may be vaccine driven in 2 MTs prevalent in Australia.

This increase of allelic frequency is better refl ected in changes in antigenic profi les. MT27 has 3 profi les (prn1, fi m2-1, fi m3-A; prn2, fi m2-1, fi m3-A; and prn2, fi m2-1, fi m3-B). The fi rst profi le was seen once in the WCV period, whereas the other 2 fi rst appeared in the WCV/ACV transition period and increased in frequency in the ACV period; the third profi le, which differed by 2 alleles from the fi rst, was more frequent. The appearance of prn2 in the second profi le and additional change from fi m3-A to fi m3-B in the third represent increases in prevalence of alleles absent from ACV. MT29 also has 3 profi les ([prn1, prn2, or prn3], fi m2-1, fi m3-A), which differ in prn only. Most MT29 isolates carry prn3, and the profi le is prevalent in both WCV and ACV periods, with no obvious increase in non-ACV alleles.
MT70 and MT64 both have uniform allelic profi les (prn1, fi m2-2, fi m3-A and prn1, fi m2-1, fi m3-A, respectively). However, MT70 (with fi m2-2, not likely to be in ACV) increased signifi cantly over the study period while MT64 with all alleles likely to be in ACV remained steady.
Overall, the frequency of MT27 and MT70, with non-ACV alleles, increased signifi cantly (p<0.0001) and correlated with the introduction of ACV, suggesting that antigenic changes could be driven by selection pressure.
The 2 MTs predominant in Australia were also prevalent in other countries and possibly have a global distribution. MT27 (18% of isolates) was found in 8 countries and MT29 (17% isolates) in 5. However, absence of an MT in a country might result from the small samples used. MT27 and MT29 were the most common types in the Netherlands (10) and the United Kingdom (11). MT10, MT64, MT70, MT84, and MT186 were also relatively common. MT10 and MT186 were found predominantly in Japan, although each had been found elsewhere, in China (1957) and Hong Kong (2002), respectively. MT64 was predominantly from Australia with 1 isolate from Japan, and MT70 was only found in Australia. However, all of these frequent MTs (except MT186) have been observed before. MT10 was frequent in the United Kingdom in the prepertussis vaccine era, while MT70 was common during 1998-2001 (11  MLVA data were used to construct a minimum spanning tree (MST) (Figure 2). The 66 MTs were grouped into 2 clonal complexes and 9 singletons. Most MTs (54 of 66) belong to 1 clonal complex and 3 (MT186, MT187, and MT194) belong to another. Relationships between singletons with multiple allelic differences are not robust because they can be connected to other nodes equally. Thus, the MST cannot be rooted to infer the direction of change. Two internationally predominant MTs (27 and 29) are closely related with 1 allele difference. MT10, prevalent in Japan, is also closely related to MT29, with 1 allele difference. MT29, fi rst isolated in the prevaccine era in the United Kingdom (11), has the highest number of SLVs and was found over 4 continents, which suggests that it arose early. Because MT10 and MT27 have a high frequency of SLVs, both likely emerged quite early. MT10 was isolated as early as 1957 in China and MT27 in 1950 in the Netherlands (10). Two high-frequency MTs (MT64 and MT70) were found in Australia only recently, with few SLVs, and may have contributed to the resurgence of pertussis in Australia.

Conclusions
Analysis of 208 isolates from Australia and representative isolates of common pulsed-fi eld gel electrophoresis types from Canada, Japan, Finland, and the United States identifi ed 6 predominant MTs (clones). Two (MT27 and MT29) were distributed worldwide, while 4 (MT10, MT64, MT70 and MT186) predominated in specifi c countries. Several MTs have persisted over long periods, including 3 that have circulated for at least half a century. Typing of genes encoding ACV antigens showed that use of ACV may have driven antigenic changes of 2 MTs now predominant in Australia.   Table 1). Two new VNTR loci named VNTR7 and VNTR8 were found to be variable in the 18 isolates tested with 2 and 3 alleles, respectively. They were added to the initial set of 6 to develop an 8-plex PCR reaction.

WCV
Because no conditions satisfied the simultaneous amplification of all 8 products consistently, the primers were divided between two 4-plex PCR assays with VNTR 1, 5, 6, and 8 in 1 reaction and VNTR 2, 3, 4, and 7 in the other. The thermal cycling parameters for the 2 assays were identical, except that the annealing temperature was 68°C for the first and 60°C for the second 4-plex PCR.
The primers and dye labels are listed in Table 1  MLVA type has a subtype in the 8-locus scheme. MLVA type 29b (2 isolates, profile 8-3-7-0-7-6-10-3-3) is a subtype of MLVA type 29 (profile 8-3-7-0-7-6-9-3-3). Therefore, MLVA types based on 5 VNTR loci are compatible with those based on 8 loci, which allows comparison with previous typing data. Although the extra 3 VNTRs do not contribute greatly to discriminatory power, their inclusion is useful because they act as confirmatory markers for some genotype divisions. MLVA type is abbreviated as MT in the main text.

Bioinformatics
A minimum spanning tree (MST) was constructed by using Bionumerics (Applied Maths, Kortrijk, Belgium) based on categorical coefficient and the eBURST (4) priority rule of the highest number of single-locus changes for the clustering. A clonal complex was defined as consisting of isolates that were single locus variants (SLVs) of the predominant MLVA type (4).
The Simpson's index of diversity (D value) was calculated by using an in-house program, the MLEECOMP package (5).