A collection of 113 epidemiologically unrelated Streptococcus agalactiae strains were studied [group B streptococcus; GBS]: they belonged to different serotypes and were isolated frompregnant women in China and Russia . The insertion sequence ISSa4was found in 21 of 113 strains [18,6%] . All of the strains withISSa4 belonged to serotypes II and II/c and were characterizedby the presence of IS1381 and IS861 as well as the absence ofIS1548 and GBSi1 . All of the strains with ISSa4 possessed bothbca and bac virulence genes coding for and ß antigens,respectively . Among 21 ISSa4-positive strains, 13 differentHindIII patterns [D1 to D13] hybridizing with an ISSa4 probewere found . One of them [D13] contained a single HindIII hybridizationfragment 6.5 kb in size that was found to be specific for allISSa4-positive GBS strains . Multiple target sites for insertionsof ISSa4 were identified and included a putative pathogenicityisland, "housekeeping" genes, and intergenic regions, as wellas the genes for hypothetical proteins . No significant similaritywas observed in the sequences of the target genes for ISSa4insertions, in the relative location of the target genes on the chromosome, or the biological functions of the encoded proteins. The possible significance of ISSa4-based differentiation ofthe strains and the presence of possible "hot spots" for insertionsof ISSa4 in GBS genome are discussed.

 
Streptococcus agalactiae [group B streptococcus; GBS] is an important human pathogen that causes significant pathology during pregnancy and in neonates [17] . GBS strains are subclassified into nine serotypes according to the immunologic reactivityof the polysaccharide capsule . Approximately one-third of GBSclinical isolates in the United States belong to serotype V[9] . GBS strains of serotypes II and III predominate in thePeople's Republic of China, and GBS strains of the serotypesVI and VIII predominate in Japan [12] . Serotype III is responsible for most cases [80%] of neonatal GBS meningitis [19].

Mobile genetic elements, such as bacteriophages, transposons,and insertion sequences [IS elements], can contribute to microbial evolution and virulence by mediating genetic recombination events[13] . Numerous reports on the presence of IS elements in GBShave been published [4, 7, 8, 11, 18, 20, 21] . Some of the ISelements were found to be integrated in virulence genes thatinfluenced the changes in virulence properties of GBS [8, 11, 20] . Following the complete sequencing of two GBS strains, severalnew IS elements were identified along with their precise locationon the chromosome [6, 22] . ISSa4, as a member of IS982 family, has been previously found by Southern hybridization in fourGBS strains and sequenced [20] . Until now, it had not been found in other bacterial species . Also ISSa4 was not found in the genomes of the two GBS strains sequenced [6, 22] . Furthermore,the nucleotide sequence of ISSa4 did not reveal any significantsimilarity to other known bacterial IS elements.

Recently we examined the distribution of IS861 and IS1548 among the collection of GBS strains and analyzed the relative locations of IS861 and IS1548 on the chromosomal DNA [4] . We could revealseveral genetic lineages of GBS based on restriction fragmentlength polymorphism [RFLP] analysis of IS861 and IS1548 andthe analysis of the chromosomal DNA by pulsed-field gel electrophoresis.The goal of the present study was to further examine the samecollection of GBS strains in order to investigate the distributionof ISSa4 among GBS strains of different serotypes and to analyzethe copy number of ISSa4 in different strains . Another goal of the study was to evaluate ISSa4-based differentiation ofthe strains and to identify the target sites for insertionsof ISSa4 copies in the GBS genome.

 
Bacterial strains. A total of 113 epidemiologically unrelated GBS strains wereanalyzed . Among them, 100 strains were isolated in differentregions of the People's Republic of China in 1996 to 2000, and13 strains were isolated in Saint Petersburg, Russia, in 1989 to 1995 . All GBS strains were isolated from the healthy pregnant women without any symptoms of streptococcal diseases . Serotyping was done with the set of type-specific antisera [Ia to VIII] from the WHO Collaborating Centers for Reference and Researchon Streptococci [Saint Petersburg, Russia, and Beijing, People's Republic of China] . Serotyping of the strains revealed different serological types [Ia, Ia/c, Ib/c, II, II/c, II/R, III, III/R,and V] . Five strains were nontypeable [Table 1] . Bacteria were grown either in Todd-Hewitt broth or on 1.5% horse blood agar at 37°C overnight.

 
General DNA techniques. Most of the molecular genetic procedures were carried out accordingto Maniatis et al . [14] . Southern hybridization of HindIII-digestedchromosomal DNA with an ISSa4 probe was accomplished with theEnzo DNA labeling and detection kit [Roche, Mannheim, Germany].PCR was carried out with 30 cycles of amplification steps of30 s at 94°C, 1 min at 47 to 49°C [Table 2] and 1 minat 72°C . Sequencing of PCR products was performed with anABI Prism 377 Perkin-Elmer sequencer and a BigDye terminatorkit [Applied Biosystems] . The primers used for cloning and sequencingare listed in Table 2.

 
Computer analysis. The sequences of the genes were accessed through the database[http://www.ncbi.nlm.nih.gov/entrez] . The sequences of the primerswere designed with the computer program OLIGO . Computer analysisof DNA sequences was accomplished by using BLAST [http://www.ncbi.nlm.nih.gov/blast]. After electrophoresis, the sizes of DNA fragments were estimated with the program SEQAID by using a 100-bp ladder [Bio-Rad Laboratories] or HindIII fragments as DNA molecular size standards.

Nucleotide sequence accession numbers. The sequence data reported in this paper appear in the GenBanknucleotide sequence database under accession numbers AY445916, AY449757, AY449758, AY449759, AY455996, AY455997, AY459524, AY461800, and AY461801.

 
Presence of ISSa4 in GBS strains. The presence of ISSa4 in 113 human GBS strains was studied byPCR . Two forward primers [105 and 150] and two reverse primers[106 and 151] were used for amplification of the different regionsof ISSa4 . As a result, 21 of 113 strains [18.6%] were foundto possess ISSa4 . Interestingly, ISSa4 occurred among the strainsof serological types II and II/c and was not found in any ofthe strains of other serotypes [Table 1].

In order to confirm the serotype-specific occurrence of ISSa4,an additional collection of 90 GBS strains of serotypes Ia,Ia/c, Ib/c, II, II/c, II/R, III, III/R, and V was analyzed.In this collection, each serotype was presented by 10 strains.As a result, ISSa4 was found in 13 of 90 GBS strains . Amongthese 13 strains, 8 strains belonged to serotype II/c and 5strains belonged to serotype II that confirms the serotype-specificoccurrence of ISSa4 . This serotype-specific occurrence explainswhy ISSa4 was not discovered during the complete GBS genomesequencing of serotypes III and V [6, 22].

The presence of several other IS elements [i.e., IS1548, IS861, and IS1381] and intron GBSi1 was determined by PCR with the primers listed in the Table 2 . Taken together, 9 different geneticvariants were discovered among 113 strains tested [Table 3].All strains of serotype II/R possessed IS1381, IS861, and GBSi1.IS1548 was found among the strains of serotypes III and III/R.Taking into account the presence of other IS elements, the strainsof serotypes III and III/R could be effectively classified intodifferent IS-based genetic variants [Table 3] . These resultsconfirm the presence of different genetic lineages among thestrains of serotypes III and III/R previously revealed by multilocusenzyme genotyping and ribotyping [1, 16] . At the same time, strains of serotypes III and III/R were found to be different from the strains of other serotypes . These results are in agreement with previous publications that demonstrated the differencebetween restriction profiles of the strains of serological typesIII and III/R in comparison with the strains of other serotypes[2, 5, 10] . The strains with ISSa4 belonged to serotypes IIand II/c and were characterized by the presence of IS1381 andIS861 as well as the absence of IS1548 and GBSi1 [Table 3].Analysis of the presence of different IS elements in GBS strainsrevealed the mutually exclusive presence of IS1548, ISSa4, andGBSi1 in the GBS genome [Table 3] . These data confirm the mutuallyexclusive presence of IS1548 and GBSi1 recently revealed [7]. It is probable that the strains with ISSa4 represent a unique genetic lineage that is different from the strains with IS1548 and the strains with GBSi1.

 
As mentioned above, the strains of serotypes III and III/R were classified into different genetic variants . The same was truefor GBS strains of other serotypes with exception of the strainsof serotype II/R [Table 3] . All of these data indicate that IS-based typing can be considered an additional tool for further differentiation of GBS strains.

Additional analyses of GBS strains for the presence of virulence genes bca and bac coding for and ß antigen, respectively,were performed [data not shown] . Recently we demonstrated thatthe presence of bca and bac encoding for and ß antigens,respectively, could be used as a marker for differentiationof GBS strains into different genetic lineages [2, 3] . In thisstudy, it was found that 21 strains with ISSa4, including 4strains of serotype II and 17 strains of serotype II/c [Table1], possessed both bac and bca genes . Some other strains ofdifferent serotypes [Ia, Ia/c, Ib/c, II, and II/c] with thebca gene or both bca and bac genes lacked ISSa4 . Taken together,these data indicate that one of the GBS clones of serotype II[II/c], with bac and bca genes as well as IS1381 and IS861, could be a recipient of ISSa4 due to the horizontal transfer from other organism.

RFLP of ISSa4. The chromosomal DNA of 21 GBS strains with ISSa4 were digestedwith HindIII and hybridized with the ISSa4 probe . The numberof HindIII fragments hybridizing with the ISSa4 probe variedsignificantly in the strains; the largest number of HindIIIfragments was 14, and the smallest number was 1 . Taken together,13 different HindIII hybridization patterns [D1 to D13] werefound among 21 strains [Fig . 1], demonstrating the good discriminativevalue for differentiation of the strains containing ISSa4.

 
In this study, it was considered that each copy of ISSa4 didnot contain a HindIII restriction site as was published forthe recently sequenced ISSa4 [20] . Based on this hypothesis, the number of ISSa4 copies could presumably correspond to the number of HindIII fragments hybridizing with the ISSa4 probe. Hybridization pattern D13 was presented by a single 6.5-kb HindIII fragment that indicated the presence of one copy of ISSa4 in the genome . This fragment was present in all other patterns[D1 to D12] and was found to be specific for GBS strains withISSa4 [Fig. 1] . This observation probably indicates that the 6.5-kb HindIII fragment contains the original target site for integration of ISSa4 in the GBS genome [Fig . 1] . The strain characterized by pattern D13 was isolated earlier than all other strains and could be considered to be the closely related to the original ancestor clone . The following duplications of ISSa4in different GBS clones occurred independently and could produceother clones with various numbers of ISSa4 copies . If true,the similarity between the ISSa4 hybridization patterns [Fig.1] can reflect the degree of genetic relationship between thestrains with ISSa4 . Nevertheless, this hypothesis should beconfirmed in future experiments.

Identification of the target sites for ISSa4 insertions. Previously, multiple copies of ISSa4 in the GBS genome were identified by Southern hybridization . However, the precise location in the genome was identified for only one ISSa4 copy . Insertionof this copy occurred in the cylB gene and resulted in inactivation of GBS hemolytic activity [20].

In the present study, the following strategy was chosen for identification of the target sites for insertions of ISSa4.The HindIII fragments of the chromosomal DNA were ligated intothe HindIII-digested pGEM-7zf[+] cloning vector . Amplificationof the ligation mixtures with primers 274 [or 275] and 106 [Table 2] produced different PCR products, some of which were isolatedfrom agarose gels, purified, and sequenced.

The sequences of the PCR products were compared with the sequences of the complete GBS genomes [6, 22] and multiple target sitesfor insertions of ISSa4 were identified [Table 4] . Target site4 [Table 4] was identified inside one of the putative pathogenicityislands previously described in GBS [6, 22] . Other target geneswere located in different parts of the genome and insertionsof ISSa4 could occur in housekeeping genes, intergenic regions,and genes for hypothetical proteins [Table 4] . Most of the targetregions identified were found to be strain specific [Table 4].These observations confirm the independence of the ISSa4 duplicationsin different GBS strains . However, the insertion of ISSa4 inthe hypothetical protein [target 5] was found among 19 of 21strains [Table 4] . It probably demonstrates that this geneticevent occurred at an early stage of evolution of the ISSa4-positiveGBS lineage.

 
Previously, insertion specificity was described for some ISsin different bacterial species [13]; however, little was known about transposition of the members of the IS982 family, including ISSa4 . In this study, no significant similarity was observedin the sequences of the target genes for ISSa4 insertions, inthe relative location of the target genes on the chromosome,or biological functions of the encoded proteins . Nevertheless, insertions of ISSa4 occurred inside AT-rich regions [Table 4], and the AT-rich regions could be considered as "hot spots" for insertions of ISSa4, as was also described for IS1 family members [15].

In summary, ISSa4-positive GBS strains belong to serotypes IIand II/c and represent a specific genetic lineage characterizedby the presence of IS1381 and IS861 as well as the virulence genes bac and bca . Copy number and the RFLP of ISSa4 can be effectively used for differentiation of GBS strains of serotypes II and II/c . No insertion specificity was observed for ISSa4; however, AT-rich regions could be considered as hot spots forISSa4 insertions.

 
We are thankful to Michael Chaussee [University of South Dakota, Vermillion] and Alexander Suvorov [Institute of Experimental Medicine, Saint Petersburg, Russia] for comments that helpedus publish this paper in the present form.

This work was supported by funding from the Ministry of Science and Technology, People's Republic of China, and by Russian President grant 2206.2003.4.

 
* Corresponding author . Mailing address: Beijing Children's Hospital Affiliated to Capital University of Medical Sciences, Nan Lishi Road, 56, Beijing 100045, People's Republic of China . Phone: 8610-6802-8401 . Fax: 8610-6801-1503 . E-mail: yyh66@vip.sina.com.

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