Capsular gene typing of Streptococcus agalactiae compared to serotyping by latex agglutination

Abstract

We evaluated three different PCR-based capsular gene typing methods applied to 312 human and bovine Streptococcus agalactiae (group B Streptococcus [GBS]) isolates and compared the results to serotyping results obtained by latex agglutination. Among 281 human isolates 27% could not be typed by latex agglutination. All 312 isolates except 5 could be typed by the three PCR methods combined. Two of these methods were multiplex assays. Among the isolates that were typeable by both latex agglutination and capsular gene typing, 94% showed agreement between the two methods. However, each of the PCR methods showed limitations. One of the methods did not include all 10 recognized serotypes, one misidentified eight isolates of serotypes Ib and IV as serotype Ia, and one did not distinguish between serotypes VII and IX. For five isolates that showed aberrant patterns in the capsular gene typing, long-range PCR targeting the cps operon disclosed large insertions or deletions affecting the cps gene cluster. A sensitive flow cytometric assay based on serotype-specific antibodies applied to 76 selected isolates that were nontypeable by latex agglutination revealed that approximately one-half of these did express capsular polysaccharide. A procedure for convenient and reliable capsular gene typing to be included in epidemiological and surveillance studies of S. agalactiae is proposed.

Fig 1

Serotype IX strains are identified as serotype VII in the two-set multiplex PCR (14). Lanes 1 to 3, amplicons from the multiplex PCR performed as described by Imperi et al. (11); lanes 4 to 6, amplicons from the multiplex PCR 2 performed as described by Poyart et al. (14). M, molecular weight marker; lanes 1 and 4, strain CZ-PW-093 (serotype VII); lanes 2 and 5, strain CZ-PW-032 (serotype IX); lanes 3 and 6, DE-PW-092 (serotype IX).

Fig 2

Examples of weak bands in the multiplex PCR performed as described by Imperi et al. (11). M, molecular weight marker; lane 1, DE-PW-096 (serotype Ib); lane 2, DE-PW-203 (serotype Ib); lane 3, DE-PW-196 (serotype IV); lane 4, ES-PW-167 (serotype IV); lane 5, DE-PW-091 (serotype Ia). The weak bands are marked by asterisks.

Fig 3

Long-range PCR with primers cpsA-F and neuA-R flanking the cps locus on the five strains that were nontypeable by the molecular capsular gene typing method. For comparison, three strains that could be typed by both serology and PCR are included. Lane 1, CZ-PW-123 (serotype Ia); lane 2, DE-PW-121 (serotype III); lane 3, DE-PW-119 (serotype Ib); lane 4, GB-PW-013 (nontypeable); lane 5, USS215 (nontypeable, bovine strain); lane 6, IT-PW-0085 (nontypeable); lane 7, IMMI266 (nontypeable); lane 8, GB-PW-075 (nontypeable); M, molecular weight marker.

Fig 4

Diagram illustrating the proposed procedure for capsular gene typing of S. agalactiae. PCR 1 and PCR 2 are the two multiplex PCRs 1 and 2 described by Poyart et al. (14) and analyzed by 1% agarose gel electrophoresis. PCR 3 is a multiplex PCR using primers cpsI-Ia-6-7-F, cpsI-7-R, cpsI-7-9-F, and cpsI-9-R and analyzed by 1.5% agarose gel electrophoresis as described by Imperi et al. (11).