EID Journal Home>Volume 16 Number 11 November 2010
Abstract:
To determine prevalence and genetic structures of new serotype 6D strains of pneumococci, author examinded isolates from diverse clinical specimens in South Korea during 1991 to 2008. Fourteen serotype 6D strains accounted for 10.4% of serogroup 6 pneumococci from blood, sputum, nasopharynx and throat samples. Serotype 6 D strains consisted of 3 sequence types.
Streptococcus pneumoniae is a common cause of invasice infection in infants, children and adults. The polysaccharide capsule of S.pneumoniae is the major virulence factor that protects the organism from host phagocytosis. Recently, 2 new serotypes of serogroup 6 pneumococci, 6C and 6D were genetically and biochemically characterized. Serotype 6C was identified in 2007 on the basis of its distinct binding patterns with 2 monoclonal antibodies; serotype 6C had previously been typed as 6A according to the standard quellung reaction.Serotype 6C produces glucose in the place of galactose in the 6A capsular polysaccharide and has the wciNbeta gene, which is about 200 bp shorter than the corresponding wciN gene in 6A. After the discovery and characterization of 6C through genetic and biochemical studies, a new experimental serotype, 6X1(later named 6D) was created by mutating the critical nucleotide in the wciP gene of the 6C capsule gene locus or by inserting the wciNbeta gene into the 6B capsule gene locus. However, this putative serotype, 6D was thought to not exist in nature until recently, when 2 studies found 6D strains in nasopharyngeal aspirates from children in Fiji during 2004 to 2007 and in 2 nasopharyngeal aspirates from children in South Korea in 2008. Although serotype 6C has only recently been described, several studies indicate that serotype 6C pneumococci have been circulating in many countries, including the US, the Netherlands, Australia, Israel and South Africa. However, reports of naturally occurring serotype 6D pneumococci are limited.
Author investigated the prevalence of serotypes of 6C and 6D in 2 collections of pneumococci isolated from clinical specimens in South Korea, we compared the genetic diversity and antimicrobial drug susceptibility patterns of the 4 serotypes, 6a, 6B, 6C and 6D.
The Study:
Of the 2 collections of pneumococcal isolates, the first consisted of 587 clinical specimens obtained from infants and children at Seoul National University Children's Hospital, Seoul, South Korea, from May 1991 to May 2008. The second collection consisted of 225 clinical specimens obtained from adults at 2 participating hospitals in Seoul from March 2004 through August 2007. When >1 isolate was recovered from the same person, only the initial isolate was included in the study. From these 2 sample collections (n=812), author redetermined serotypes for 134 isolates previously assigned to serogroup 6.
Serotyping was performed by uisng the quellung reaction with antiserum for serogroup 6, factor 6b and factor 6c(Statens Serum Institute,Copenhagen, Denmark).To assign serotypes 6C and 6D we screened all strain for wciNbeta and wciP-6B by using two simple PCRs and subsequent sequencing analysis. The wciN gene was amplified with the forward primer (5106) 5-TAC CAT GGA GGG TGG AAT GT-3 and reverse primer (3101) 5-CCA TCC TTC GAG TAT TGC-3, resulting in product size of 1.8 kb for serotypes 6C and 6D for the wciNbeta gene. The wciP gene was amplified by using the forward primer 5-AAT TTG TAT TTT ATT CAT GCC TAT ATC TGG-3 and the reverse primer 5-TTA GCG GAG ATA ATT TAA AAT GAT GAC TA-3. Presence of wciNbeta and wciP-6B was confirmed by sequencing analysis. A characteristic of 6B wciP is the presence of an A at nucleotide position 584 (according to the sequence of wciP), which creates a codon for asparagine at residue 195 of the 6B wciP protein. Antimicrobial drug susceptibility testing, multilocus sequence typing -MLST and eBURST analyses were performed.
Conclusions:
We identified 14 naturally occuring serotype 6D strains among 134 serogroup 6 pneumococci collected from diverse clinical specimens in South Korea during 1991 to 2008. The prevalence rate of serotype 6D among serogroup 6 isolates was 10.4%, slightly higher than that of serotype 6C(4.5%). Although serotype 6D was only recently discovered, we demonstrated that serotype 6D strains have been circulating since at least 1996. Serotype 6D was identified from various clinical sources, including blood, sputum, throat swab and nasopharynx specimens, contrasting with findings of 2 previous studies.
The genetic structures of serotype 6D pneumococci in the MLST database http://www.mlst.net/ were single isolates o f ST4241(Australia); ST982, ST4190, ST5085 and ST5086 (China) and 2 isolates of ST282 (South Korea). Of those, 3 strains from China (ST982, ST5085 and ST5086) were closely related to the ST3171 strain from South Korea. This cluster of serotype 6D strains was associated with serotype 6A and 6B isolates from 3 countries in Asia. A single isolate of ST4241 was related to STs associated mostly with serotype 6B, but the ST4170 strain did not seem to be linked to other STs. This study demonstrated that 7 serotype 6 D strains of ST189 and 3 serotype 6D stains of ST282 were related to clonal complex 81, which had previiously been associated with many other global serotypes, such as 23F, 19F and 19A. Although the mechanism is not completely clear, available data indicate that capsular switching from serotypes 6A, 23F, 19F or 19A to serotype 6D is possible, this switching could occur in addition to replacement of the wciNbeta gene into the 6B capsule gene locus. A previous study indicated capsular switching as the possible event for formation of serotype 6C isolates.
In a recent study, factor 6D antiserum was validated for accurate serotyping of 6C and is now commercially available, but antiserum for detection of 6D has not yet been developed. Further studies will be required to investigate the prevalence and genetic relatedness of serotype 6D pneumococci in different countries and to evaluate the effect of pneumococcal conjugate vaccine on serotype distribution.
QUELLUNG REACTION:
The Quellung reaction is a biochemical reaction in which antibodies bind to the Bacterial capsule of Streptococcus pneumoniae, Klebsiella pneumoniae, Neisseria meningitidis and Haemophilus influenzae and thus allow them to be visualized under a microscope. If the reaction is positive, the capsule becomes opaque and appears to enlarge.
Quellung is the German word for "swelling" and describes the microscopic appearance of pneumococcal or other bacterial capsules after their polysaccharide antigen has combined with a specific antibody. The antibody usually comes from a bit of serum taken from an immunized laboratory animal. As a result of this combination, and precipitation of the large, complex molecule formed, the capsule appears to swell, because of increased surface tension, and its outlines become clearly demarcated.
The pneumococcal Quellung reaction was first described in 1902 by the scientist Fred Neufeld, both as microscopic capsular swelling and macroscopic agglutination (clumping visible with the naked eye). It has been used to identify the 90 known capsular serotypes of Streptococcus pneumoniae in a diagnostic setting, but in recent years has been challenged by molecular typing techniques which target genetic differences.
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