Typing of Streptococcus agalactiae isolates using DNADNAmicroarrays Heike Nitschke (1) Institute
(1),
Peter Slickers (2), Kristina Hochauf (1), Florian Gunzer (1), Ralf Ehricht (2), Stefan Monecke (1)
for Medical Microbiology and Hygiene, Faculty of Medicine “Carl Gustav Carus”, Technical University of Dresden (2) CLONDIAG GmbH, Germany
Streptococcus agalactiae or group B streptococcus (GBS) is frequently carried in the normal vaginal flora, but it is also a common cause of neonatal sepsis and meningitis. This pathogen has several different virulence-associated markers which might be used to assess its ability to cause invasive disease. In order to obtain information on all these markers within a single experiment, a microarray was developed and tested on sequenced strains and clinical isolates. The capsular gene cluster, the alleles of the gene encoding the alpha-like protein alp, the variants of the quorum sensing gene cluster rgf, the presence or absence of the gene encoding the beta antigenic cell wall protein as well as absence or presence and variation of two gene clusters encoding pili can be simultaneously assayed. These markers provide a framework to split Streptococcus agalactiae in a number of strains or clusters since they vary independently from each other. Approximately 240 isolates and reference strains tested so far have been assigned to some 20 of these strains or clusters. The correlation to MLST (multilocus sequence typing) is currently under investigation. Comparative screening of clinical and surveillance isolates is planned in order to identify predictors for increased virulence among gynaecological S. agalactiae isolates and to assess the risk of invasive infection in newborn infants. Introduction: Streptococcus agalactiae or group B streptococcus (GBS) belongs to the family of Streptococcacae. GBS are gram-positive, aerotolerant and grow in pairs or chains. They are frequently carried in the normal vaginal flora, but they are also a common cause of neonatal sepsis and meningitis (early onset as well as late onset). S.agalactiae has several different virulence-associated markers which might be used to assess its ability to cause invasive disease. In order to obtain information on all these markers within a single experiment, a microarray was developed and tested on sequenced strains and clinical isolates. Methods: Vaginal swabs were collected as part of routine antenatal screening. Blood cultures were obtained for diagnostic purposes in cases of suspected neonatal sepsis/meningitis (courtesy of R. Berner, Freiburg). S. agalactiae were sub-cultured on Columbia blood agar. For nucleic acid preparation, culture material was lysed using a buffer containing ribonuclease, lysozym and achromopeptidase. Then, the lysate was transferred into spin columns (QIAamp DNA Mini Kit, QIAGEN, Hilden, Germany). After preparation, DNA concentration was determined. The target DNA was labelled with biotin-dUTP by performing a linear primer elongation reaction. For this reaction, a primer mixture was used which contained one primer for each of the 213 target sequences. Labelled amplicons were diluted in hybridisation buffer and hybridised with probes on the DNA-arrays (based on the CLONDIAG ArrayStripe system). After hybridisation, conjugation and incubation with horse-radish-peroxidase substrate, hybridisations were detected by adding a precipitating dye. Images of the array were taken with the ARRAYMATE device by CLONDIAG. Array images were converted in strings or ‘sequences’ using ‘A’ for positive and ‘T’ for negative results. These datasets were used for tree construction using SPLITSTREE 4 (Huson & Bryant, 2006) software (characters transformation, uncorrected P; distance transformation, Neighbour-Net; and variance, ordinary least squares). Thus it was possible to obtain a split network tree which was used to visualise similarities between hybridisation patterns. Some selected isolates with representative hybridisation patterns were subjected to multilocus sequence typing (MLST). This was performed using previously published protocols (see http://pubmlst.org/sagalactiae/) using amplification and sequencing primers for the genes adhP, atr, glnA, glcK, pheS, sdhA and tkt. The amplification products were purified on invisorb columns (invitek, Berlin, Germany) and sequenced with an automated DNA sequencer (Applied Biosystems, Darmstadt, Germany).
Figure 1: DNA microarray images of a sequenced reference strain (COH1) and three different clinical isolates.
Results The capsular gene cluster, the alleles of the gene encoding the alpha-like protein alp, the variants of the quorum sensing gene cluster rgf, the presence or absence of the gene encoding the beta antigenic cell wall protein as well as absence or presence and variation of two gene clusters encoding pili were simultaneously assayed. These markers provided a framework to split S. agalactiae in a number of strains or clusters since they vary independently from each other (see Figure 2). 240 clinical isolates and sequenced reference strains (COH1, A909, 515, H36B, 18RS21, CJB 111, 2603VR, CIP 8245) have been tested so far. Hybridisation patterns for sequenced reference strains were in accordance to predictions derived from their published genome sequences. For some clinical isolates, MLST is currently performed. Preliminary data show that hybridisation patterns correspond to sequence types as defined by MLST. However, only complete hybridisation profiles but no single markers were characteristic for a sequence type. It was observed that carriage as well as invasive isolates occurred in all sequence types or in all clusters as defined by hybridisation patterns, respectively. However, a considerable number of invasive isolates belonged to capsule type III and carried the rib (R4) allele of the gene encoding the alpha-like protein. Figure 2: Population structure of S. agalactiae based on array hybridisation patterns. Sequenced reference strains, multilocus sequence types, capsule types and alleles of the gene encoding the alpha protein are indicated.
Discussion and conclusions With the DNA microarray technology it is possible to genotype clinical isolates of various bacteria such as S. agalactiae with regard to the carriage of virulence factors and to the affiliation to phylogenetic units. Similar as previously shown for S. aureus, these units appear to correspond to sequence types and clonal complexes as defined by MLST. We observed that otherwise similar stains might differ in single features such as capsule genes, genes encoding cell wall proteins as well as the pili gens, and that these genes might vary independently from each other. This could be explained either by past recombination events, or by a convergent evolution leading to a limited number of, e.g., capsule types. DNA microarray technology allows rapid assigning of large numbers of clinical isolates without the need for sequencing. An interesting objective for an array-based study is to compare GBS isolates from healthy carriers with isolates obtained from cases of invasive disease. First data show that there was no fundamental difference between GBS populations from carriage and patient samples. However, one cluster (capsule type III/alp-rib (R4)) appears to be over-represented among cases of invasive disease. This warrants further investigations.
Acknowledgments: The authors thank R. Berner (Center for Pediatrics and Adolescent Medicine, University Medical Center Freiburg) and the colleagues of the Clinic for Gynaecology and Obstetrics, Dresden University Hospital for providing samples as well as A. Ruppelt, E. Müller, I. Engelmann, J. Sachtschal, G. Rößler and the staff of Bacteriological laboratory of the Institute for Medical Microbiology and Hygiene for excellent technical assistance. We acknowledge Prof. E. Jacobs and E. Ermantraut for their support. The study was funded by the Vice-Directorate for Research of the Faculty of Medicine “Carl Gustav Carus” (MedDrive program 2009).
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