Pneumococcal pspA sequence types of prevalent multiresistant pneumococcal strains in the United States and of internationally disseminated clones

Abstract

In a recent genotypic survey of beta-lactam-resistant pneumococci recovered in different areas of United States during 1997, eight clonal types that each represented 3 to 40 isolates accounted for 134 of 144 isolates (G. Gherardi, C. Whitney, R. Facklam, and B. Beall, J. Infect. Dis. 181:216-229, 2000). We determined the degree of pspA gene diversity among these 134 isolates and for 11 previously characterized internationally disseminated multiresistant strains. Thirty-four different pspA restriction profiles were determined for an amplicon encompassing the variable portion of the structural gene that encodes the surface-exposed domain of PspA and a variable-length proline-rich putative cell wall-associated domain. These restriction profiles closely correlated with those of 33 different pspA sequence types of an approximately 230-residue region corresponding to residues 182 to 410 of the strain Rx1 PspA. These residues encompass a 100-residue clade-defining region known to contain cross-protective epitopes for which 17 sequence types were found. Distinct, conserved pspA sequence types were found for the majority of strains within seven of the eight U.S. clonal types assessed, while one pulsed-field gel electrophoresis type was represented by isolates of three distinct PspA clades. Sequence typing of pspA provides an added level of specificity in the subtyping of isolates and is a necessary first step in determining the components needed in a PspA vaccine which could elicit effective cross-protective coverage.

FIG. 1

General features of PspA and method used for sequence typing of pspA amplicons. The pspA codon positions are taken from references and and are based upon the Rx1 pspA sequence (27). Abbreviations: SS, signal sequence; CDR, clade-defining region containing cross-protective epitopes; VLP, variable-length proline-rich repeat region.

FIG. 2

PspA clades of previously determined PFGE types (9) of multiresistant, invasive pneumococcal isolates. The dendrogram gives underestimated Dice coefficients because closely migrating bands could not be properly resolved with the program used. Visually identical PFGE profiles gave average Dice coefficients of ∼0.86 to 0.93. UPGMA, unweighted pair group method with arithmetic averages. The strains with the closest matches in their PspA clade-defining regions (11) are listed along with their pspA GenBank accession numbers.

FIG. 3

Alignment of the PspA clade-defining regions that contain cross-protective epitopes from different pspA amplicon RFLP types. BG6692 and DBL1 refer to PspA protein sequences from reference . The PileUp program was used as described previously (11). Positions shared by nine or more PspA sequences are shaded.

FIG. 4

RFLP patterns of pspA amplicons. Amplicons generated with primers F and R were subjected to triple digestion with HaeIII, HinfI, and RsaI prior to agarose gel electrophoresis. The RFLP type designations at the top are from Table 1. Lanes 1, 17, and 31, size ladder (L); lanes 2 to 16 and 18 to 30, 1997 multiresistant U.S. isolates; lanes 32 to 42, internationally disseminated multiresistant strains: lane 32, Spain^23F-1; lane 33, Spain^6B-2; lane 34, France^9V-3; lane 35, Tennessee^23F-4; lane 36, Spain¹⁴-5; lane 37, S. Africa^19A-7; lane 38, S. Africa^6B-8; lane 39, England¹⁴-9; lane 40, Hungary^19A-6; lane 41, Slovakia¹⁴-10; lane 42, Slovakia^19A-11.

FIG. 5

Alignment of PFGE type E (subtypes E1 to E9) PspA variable-length proline-rich regions.