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J Clin Microbiol. Nov 2006; 44(11): 4252–4254.
Published online Sep 27, 2006. doi:  10.1128/JCM.01533-06
PMCID: PMC1698356

Identification of a Streptococcus agalactiae Serotype III Subtype 4 Clone in Association with Adult Invasive Disease in Hong Kong[down-pointing small open triangle]

Abstract

Characterization of Streptococcus agalactiae serotype III isolates revealed a subtype 4 clone that has an indistinguishable pulsed-field gel electrophoresis pattern and possesses a C-α protein, IS1381, and a novel sequence type (ST), ST 283, by multilocus sequence tagging. This clone was significantly associated with diseases caused by invasive strains from nonpregnant adults (P ≤ 0.01, chi-square test) and was not present in the genital tracts of pregnant mothers.

Streptococcus agalactiae (group B streptococcus [GBS]) is a major pathogen in neonatal infections. However, an increasing number of diseases caused by GBS nonpregnant-adult-invasive strains has been observed (4, 8, 20). Serotype III is a predominant serotype causing neonatal and invasive GBS diseases in the United States, Europe, and Asia. Serotype III isolates have been classified into 4 subtypes (1 to 4) according to sequence variation in polysaccharide genes (14). Subtypes 1 and 2 correspond to sequence types (STs) 19 and 17 by multilocus sequence typing (MLST) (6, 16) and have been associated with adult endocarditis and neonatal meningitis (2, 9), respectively. The other two subtypes, 3 and 4, are uncommon (6, 14).

GBS meningitis in adults has been reported in Southeast Asia, notably in Singapore and Hong Kong (5, 20). The associated GBS strains belonged to serotype III. Jones et al. (12) demonstrated that GBS ST 11 accounted for five cases of adult meningitis from Singapore. Other studies have utilized the profiles of surface protein genes and mobile genetic elements to link the relationships of molecular serotypes and their lineages (6, 15, 16). These surface proteins have also been described to be associated with virulence (17-19). We thus sought to characterize GBS serotype III isolates from Hong Kong by subtyping of the isolates (subtypes 1 to 4) by PCR and sequencing, pulsed-field gel electrophoresis (PFGE), the detection of surface protein genes and mobile genetic elements, and MLST.

(These data were presented at the 16th ECCMID, Nice, France, 1 to 4 April 2006 [abstract no. P904]).

One hundred thirty-six nonduplicate GBS serotype III isolates, previously serotyped by the agglutination method by using 9 antisera (Denka Seiken, Japan) (10), were examined. These strains were isolated from patients admitted to the Prince of Wales Hospital, a 1,350-bed teaching hospital in Hong Kong, between 1993 and 2003. Seventy-three isolates were from sterile body sites, including blood, cerebrospinal fluid, continuous ambulatory peritoneal dialysis fluid, and joint aspirate, considered “invasive.” GBS isolated from “noninvasive” sites included 15 from wound/pus samples and 48 from swabs from newborns/genital tracts of pregnant mothers.

Eight hundred base pairs of the capsular polysaccharide genes from cpsE to cpsG were sequenced for subtyping (14). The primers and PCR conditions for the detection of the surface protein genes bca (encoding a C-α protein) and bac (encoding a C-β protein), alp2 and alp3 (encoding C-α-like proteins 2 and 3, respectively), bca-rep (encoding a C-α-repeating unit/-repeating-unit-like unit), and rib (encoding the Rib protein) and of the mobile genetic elements ISSag1, ISSag2, IS1548, IS861, IS1381, ISSa4, and GBSil were as described previously (15, 16). PFGE was performed (1) with an additional step for mutanolysin (40 U/ml) digestion. MLST was performed, and the STs of representative strains of each subtype were identified using a method described previously (12). The distribution of invasive isolates was calculated by the chi-square test.

The distribution of serotype III subtypes from neonates and pregnant and nonpregnant adults is given in Table Table1.1. The numbers of isolates from “invasive” and “noninvasive” sites are stated. Seventy-one isolates (52.2%) belonged to subtype 1, 34 isolates (25.0%) to subtype 2, 7 isolates (5.1%) to subtype 3, and 24 isolates (17.7%) to subtype 4. Subtype 4 strains were isolated predominantly from “invasive” sites (P ≤ 0.01, chi-square test) and from nonpregnant adults only. The MLSTs, protein gene profiles, and mobile genetic elements for representative isolates of subtypes 1 to 4 are listed in Table Table22 . The commonest subtype was subtype 1, and these isolates were characterized by the presence of the Rib protein gene, IS861, IS1381, and IS1548 and belonged to ST 19. Previously, IS1548-carrying strains have been significantly associated with diseases caused by adult-invasive strains, especially with endocarditis (2). However, over half of our strains were noninvasive and thus do not support the use of IS1548 as a marker for adult endocarditis. Besides, 15 of the 32 invasive cases were in neonates/children. IS1548 may be a marker of subtype 1 strains, as it was not present in other subtypes. Subtype 2 isolates belonged to ST 17 and carried the Rib protein gene, IS861, and GBSi1. Previously, the presence of GBSi1 in the GBS isolates had been associated with neonatal meningitis (9). In this study, most of these isolates were from cases of neonate-invasive strains (N = 11) and further support enhanced invasiveness of ST 17 in neonates (13). Subtype 3 included seven isolates, and most had C-α-repeating units and C-α-like protein 2 genes.

TABLE 1.
Distribution of serotype III subtypes, as isolated from “invasive” versus “noninvasive” sites in neonates and pregnant and nonpregnant adults
TABLE 2.
Protein gene and IS profiles and MLSTs for representative isolates of GBS serotype III subtypes 1 to 4a

Subtype 4 strains form a unique clone that was characterized by the C-α and C-α-repetitive sequences and the IS1381 mobile genetic element and has an indistinguishable PFGE pattern. These strains have been allocated a novel sequence type, ST 283 (9-5-7-1-3-3-2), and are single-locus variants of ST 160 (9-5-6-1-3-3-2) and ST 11 (9-3-7-1-3-3-2), according to the MLST website (http://pubmlst.org/sagalactiae/). Previously, adult-invasive strains that caused five cases of diseases in Singapore were found to belong to ST 11 (12). The majority of our isolates were from nonpregnant adults with diseases caused by invasive strains, and some had been associated with the outbreak of GBS meningitis in adults in Asia during 1998 (20). This raises the postulation of a closely related clone with invasive potential for meningitis and the possibility of a wider distribution of these strains across Asian countries. In fact, the subtype 4 noninvasive isolates (Table (Table1)1) were from adults diagnosed with cellulitis, osteomyelitis, spondylitis, and mycotic aneurysm, respectively. This reaffirms the invasive potential of this clone and brings into question the original source, as similar strains had not been identified in rectal/vaginal samples from pregnant mothers. ST 17, associated with diseases caused by neonate-invasive strains, has been shown to have arisen from a bovine ancestor (3), raising the postulation of similar derivations. Presently, the presence of the C-α protein gene and the absence of GBSi1 and IS1548 may be used as markers to identify these strains.

In Hong Kong, the majority of serotype III isolates belonged to subtypes 1 and 2. A subtype 4 clone belonging to ST 283, associated with diseases caused by invasive strains in nonpregnant adults, was identified. Closely related GBS strains belonging to ST 283 and ST 11 were likely to have accounted for the cluster of meningitis cases among nonpregnant adults in Asia (20). Other series of GBS meningitis cases in adults have been extensively reviewed (7), but their GBS isolates had not been characterized in detail. The four mobile genetic elements IS1548, IS861, IS1381, and GBSi1 may be used to distinguish these four subtypes and support the different lineages of GBS serotype III.

Acknowledgments

The work described in this paper was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region (project no. CUHK, direct grant no. 4450025).

We acknowledge the use of the Streptococcus agalactiae MLST website (http://pubmlst.org/sagalactiae/), developed by Man-Suen Chan and Keith Jolley at the University of Oxford (11). The development of this site was funded by the Wellcome Trust.

Footnotes

[down-pointing small open triangle]Published ahead of print on 27 September 2006.

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