Molecular Characterization of Nonhemolytic and Nonpigmented Group B Streptococci Responsible for Human Invasive Infections

Abstract

Group B Streptococcus (GBS) is a common commensal bacterium in adults, but is also the leading cause of invasive bacterial infections in neonates in developed countries. The β-hemolysin/cytolysin (β-h/c), which is always associated with the production of an orange-to-red pigment, is a major virulence factor that is also used for GBS diagnosis. A collection of 1,776 independent clinical GBS strains isolated in France between 2006 and 2013 was evaluated on specific medium for β-h/c activity and pigment production. The genomic sequences of nonhemolytic and nonpigmented (NH/NP) strains were analyzed to identify the molecular basis of this phenotype. Gene deletions or complementations were carried out to confirm the genotype-phenotype association. Sixty-three GBS strains (3.5%) were NH/NP, and 47 of these (74.6%) originated from invasive infections, including bacteremia and meningitis, in neonates or adults. The mutations are localized predominantly in the cyl operon, encoding the β-h/c pigment biosynthetic pathway and, in the abx1 gene, encoding a CovSR regulator partner. In conclusion, although usually associated with GBS virulence, β-h/c pigment production is not absolutely required to cause human invasive infections. Caution should therefore be taken in the use of hemolysis and pigmentation as criteria for GBS diagnosis in routine clinical laboratory settings.

FIG 1

Distribution of NH/NP GBS clinical isolates. Percentages and numbers of clinical GBS strains according to the age of the patient (A), the strain invasiveness (B), and the capsular serotype (C) for the whole collection and for NH/NP strains.

FIG 2

Phenotypes and genotypes of NH/NP clinical isolates. (A) Hemolytic and pigmentation phenotypes of the 34 GBS clinical isolates selected for sequence analysis, organized according to the identified mutation, and of GBS wild-type strains NEM316, BM110, 2603V/R, 515, H36B and A909. TH, Todd-Hewitt agar; Gr, Granada agar; Bl, Columbia agar supplemented with 5% horse blood. (B) Structure and regulation of the cyl operon. Red crossed line, transcriptional repression; green straight arrow, activation; black bent arrow, initiation of transcription. (C) Distribution of identified mutations in the cyl operon and in the cyl regulator gene abx1 among 31 GBS strains; note that no mutation was associated to the NH/NP phenotype among 3 sequenced strains.

FIG 3

Hemolytic and pigmentation phenotypes in GBS mutants. (A) Effects of deletion of the IS1381 element from the CCH1084 strain and of the insertion of the same element at the cognate locus in the NEM316 strain. (B) Effects of the genetic complementation of clinical strains mutated only in abx1 with an empty vector (+ pTCV) and the same vector containing a wild-type copy of abx1 (+ abx1). (C) Pigmentation and hemolysis of the NEM316 reference strain and corresponding in-frame deletion mutants ΔcylG, ΔcylAB, ΔcylE, ΔcylI, ΔcylX, ΔcylD, ΔcylJ, and ΔcylK. Bacterial growth, hemolysis, and pigmentation were observed on Todd-Hewitt agar (TH), Granada agar (Gr), and blood agar (Bl), respectively.