Département de Microbiologie Médicale et Moléculaire, EA 3250, Unité de Bactériologie, Centre Hospitalier Universitaire Bretonneau, 37044 Tours Cedex 01, France1
Author for correspondence: Roland Quentin. Tel: +33 2 47 47 80 56. Fax: +33 2 47 47 38 12. e-mail: quentin{at}med.univ-tours.fr
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ABSTRACT |
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Keywords: neonatal meningitis, molecular analysis, RAPD
Abbreviations: CNS, central nervous system; MEE, multilocus enzyme electrophoresis; RAPD, randomly amplified polymorphic DNA
The GenBank accession numbers for the sequences reported in this paper are AF302130 (for the 2·4 kb amplified fragment) and AF302131 (for the 2·6 kb amplified fragment).
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INTRODUCTION |
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Serotyping and molecular characterization studies of S. agalactiae isolates from various anatomical sites have suggested that only a limited number of strains from the vagina can cause neonatal meningitis. In the United States, over two-thirds of the cases of S.-agalactiae-related neonatal diseases are caused by strains that produce the type III antigen (Baker & Edwards, 1995 ). In some countries, other serotypes are frequently isolated from patients with early-onset disease (Geslin et al., 1992
). Molecular genetic studies have demonstrated that there are two major phylogenetic lineages of S. agalactiae (Helmig et al., 1993
; Musser et al., 1989
; Quentin et al., 1995
). In addition, most of the strains responsible for neonatal meningitis are non-randomly distributed in these two phylogenetic groups (Musser et al., 1989
; Quentin et al., 1995
). Therefore, the different clones of S. agalactiae strains must have different virulence attributes. However, no molecular data are currently available to support this conclusion.
An insertion sequence (IS1548) has been discovered in the hylB gene, which encodes hyaluronate lyase, of a group of S. agalactiae strains isolated from patients with endocarditis (Granlund et al., 1998 ) and in a homogeneous subgroup of isolates identified by multilocus enzyme electrophoresis (MEE). This sequence identified strains able to invade the central nervous system (CNS) of neonates (Rolland et al., 1999
). Nevertheless, many other strains isolated from the cerebrospinal fluid of neonates with meningitis do not possess this insertion sequence.
Many of the current strategies used to prevent neonates from becoming infected with S. agalactiae at birth are based on treating all women who provide positive lower vaginal or anorectal cultures with intrapartum chemoprophylaxis by intravenous administration of penicillin G or ampicillin (Baker, 1997 ). These approaches may have adverse impacts; for example, they may lead to the emergence of resistant pathogens and some patients have serious allergic reactions to penicillin. Therefore, it is important to seek genomic particularities that characterize invasive groups of strains. This will enable us to clarify the molecular mechanism of invasion of this subgroup of S. agalactiae strains and to recognize strains in the genital flora that are likely to cause invasive diseases. This may also help obstetricians to limit the use of prophylaxis and, therefore, its adverse effects.
We previously carried out randomly amplified polymorphic DNA (RAPD) experiments to assess the ability of 29 different oligonucleotide primers to characterize 114 S. agalactiae strains of various anatomical origin. One primer (5'-AgggggTTCC-3') generated a 2·4 kb fragment significantly associated with strains belonging to one of the two major lineages of the S. agalactiae species (Chatellier et al., 1997 ). Therefore, this fragment may have specific genetic features that account for its significant association with the phylogenetic distribution of strains within the S. agalactiae species. In this study, we used the same collection of strains as above to characterize this 2·4 kb fragment and to determine whether its composition contributes to this specific association. This approach identified two tRNA gene clusters flanking the 3' end of the rRNA operons in S. agalactiae. Specific combinations of tRNA genes were found to be associated with the two major lineages of S. agalactiae, and these could be used to identify two of the three virulent intraspecies subgroups of strains able to cause neonatal meningitis. Based on this analysis, we designed PCR primers to determine whether given S. agalactiae strains have a high probability of belonging to a virulent subgroup of organisms. In addition, this work illustrated that differential population analysis of bacterial genomes characterized by molecular epidemiological methods may be used to identify new genetic particularities within intraspecies variants of pathogens.
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METHODS |
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Isolation and cloning of the 2·4 kb RAPD fragment.
The 2·4 kb fragment, amplified by RAPD using the 5'-AgggggTTCC-3' primer (Chatellier et al., 1997 ), was subjected to electrophoresis in a 1·4% low-melting-point-agarose (FMC) gel containing ethidium bromide (1 mg l-1). The fragment was purified from the agarose by incubating the gel at 65 °C for 15 min, followed by two phenol extractions (v/v) and a chloroform extraction (v/v). DNA was precipitated with 0·1 M ammonium acetate and absolute ethanol. The precipitated DNA was air-dried and resuspended in 10 µl double-distilled water. The DNA fragment was inserted into the pCRII vector (TA cloning kit, Invitrogen). The fragment was partially sequenced at the 5' and 3' ends with universal primers SP6 and T7 (both from Eurogentec).
PCR.
A number of different primers were designed, based on known sequences (Table 1). A 20 µl PCR mixture was prepared as follows: PCR buffer (10 mM Tris/HCl, 50 mM KCl, 1·5 mM MgCl2; pH 8·3); 0·1 mM of each dNTP (Boerhinger Mannheim); 0·5 µM primers (Eurogentec) (Table 1
); 0·5 U Taq polymerase (Perkin Elmer); 25 ng of template DNA, prepared as previously described (Chatellier et al., 1997
). The cycling conditions were as follows: initial denaturation at 94 °C for 30 s, followed by 30 cycles at 94 °C for 15 s, 55 °C for 15 s and 72 °C for 15 s (additional 15 s at 72 °C for the last extension). The products were separated in a 1% agarose gel in TBE buffer (8·9 mM Tris, 8·9 mM boric acid, 0·25 mM EDTA; pH 8·0). Amplified products contained within the gel were stained with ethidium bromide and visualized by UV transillumination. A 1 kb ladder (Life Technologies) was used as a molecular size standard. The negative control consisted of a reaction in which the DNA template was replaced with double-distilled water. The DNA fragments were subsequently sequenced.
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RESULTS |
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Pattern P1 was significantly associated with the position of S. agalactiae strains in MEE phylogenetic division I (38 P1 strains versus three P2 strains in division I, Table 3), and pattern P2 was significantly associated with division II strains (21 P1 strains versus 52 P2 strains in division II, Table 3
) (P<10-4). In addition, pattern P1 distinguished five of the six ET11 strains, but it distinguished only 16 of the 67 strains belonging to ET12 or non-invasive subgroups. Therefore, pattern P1 appears to be a good marker for two of the three subgroups (division I and ET11) of isolates able to invade the CNS of neonates (P<10-4) (Table 3
).
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DISCUSSION |
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Our results link tRNA genes to some virulent phylogenetic subgroups of S. agalactiae. This is related to the composition of the tRNA gene clusters located at the 3' end of rRNA operons rather than to tRNA modification. Indeed, the rRNA operons of the two virulent clones of S. agalactiae strains (phylogenetic division 1 and ET11) always seem to be flanked by tRNA gene cluster 1. In contrast, rRNA operons of the other genetic groups of strains were flanked by either of the two types of tRNA gene clusters. As tRNAs are a central interface between the information stored in DNA and its expression in proteins, and as tRNA gene cluster 1 was the only cluster associated with two of the three virulent clones of S. agalactiae strains, it is possible that this tRNA gene cluster is involved in the synthesis of proteins responsible for bacterial pathogenicity. Nevertheless, tRNA gene cluster 1 is also present in the genome of non-invasive subgroups of bacteria but, in these strains, some of the rRNA operons are flanked by tRNA gene cluster 2. Therefore, if tRNA gene cluster 1 is involved in the expression of virulence factors, the smaller number of copies of this tRNA gene cluster in strains that also contain tRNA gene cluster 2 may result in there being fewer proteins involved in virulence.
Whatever the link between tRNA gene cluster composition and virulence in S. agalactiae strains, the specificities described here may be used to identify (by PCR with primers SP6-B2 and T7-B2, Table 1) isolates belonging to two of the three genetic groups of strains that can invade the CNS of neonates (division I and ET11). In addition, as we have shown previously, strains belonging to ET12, the third genetic subgroup of strains able to invade the CNS of neonates, are significantly marked by IS1548 in the hylB gene (Rolland et al., 1999
). Therefore, there is a high probability that strains that exhibit pattern P1 or contain IS1548 belong to one of these three subgroups (55 of 62 strains) rather than to the non-invasive subgroups (16 of 52 strains) (P<10-4) (Table 3
). These two characteristics may be detected by PCR with primers SP6-B2 and T7-B2, as described here, or with primers Hyal1 (5'-CAgCCACTCATAgCACAATgAAACAAg-3') and Hyal2 (5'-gCTAgTTAgATAgCTAATTgTCTgTT-3'), as described previously (Rolland et al., 1999
). These two pairs of primers may provide a valuable basis for the development of a multiplex PCR method for identifying virulent isolates among the strains present in the genital tract of pregnant women. PCR appears to be a more suitable method than RAPD for developing tools, as the interlaboratory reproducibility of RAPD remains unclear (Saunders et al., 2001
). In addition, PCR with the primer pair SP6-B2 and T7-B2, as performed in this work, identifies five of the six strains belonging to the virulent clone ET11; these strains were not identified with the 2·4 kb RAPD fragment.
By contrast, differential display of DNA following RAPD seems to be a suitable method for detecting some new genetic specificities within some subgroups of bacteria, as has been suggested previously (Hacker et al., 1997 ). The identified sequences may be phylogenetic markers, pathogenicity islands or genetic elements that play a role in cell physiology or in the expression of virulence factors. RAPD is a quick and inexpensive method, making it possible to use many primers to explore many parts of the genome for bacterial populations composed of numerous strains. This method may be as effective as subtraction hybridization for the identification of new genetic elements involved in the physiology or pathogenicity of bacteria.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 21 September 2001;
revised 16 January 2002;
accepted 21 January 2002.
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