Macrorestriction analysis of Streptococcus agalactiae (group B Streptococcus) isolates from Malaysia

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1 Journal of Medical Microbiology (004), 5, DOI 0.099/jmm Macrorestriction analysis of Streptococcus agalactiae (group B Streptococcus) isolates from Malaysia Kwai-Lin Thong, Goh Yee Ling, Leong Wing Kong, Lim Chin Theam and Yun Fong Ngeow 4 Correspondence Kwai-Lin Thong thongkl@um.edu.my Received 9 July 00 Accepted 9 June 004 Institute of Biological Sciences, Faculty of Science, Departments of OBGYN, Paediatrics and Medical Microbiology 4, Faculty of Medicine, University of Malaya, 5060, Kuala Lumpur, Malaysia Streptococcus agalactiae or group B streptococci (GBS) often colonize the gastrointestinal and urogenital tracts of women, who may transmit these organisms to their offspring during the birth process. Using PFGE analysis, the genetic diversity of GBS was studied for strains isolated from pregnant women and their newborn infants in a teaching hospital. A total of 48 different PFGE profiles were obtained from strains, with one profile (S) appearing to be predominant among both groups studied. There was good overall correlation between the profiles obtained for strains from mother infant pairs and for strains isolated from different body sites in the same individual. Occasional discrepancies seen in related body sites and among mother infant pairs suggest concurrent carriage of different strains in the same individual as well as the possibility of an environmental source of organism for the neonate. The overall results demonstrated that many variants of GBS strains occur in Malaysia. INTRODUCTION Group B streptococci (GBS) are an important cause of morbidity and mortality among neonates. In adults, these bacteria are associated with a variety of septic diseases but are also frequent colonizers of the gastrointestinal and genital tracts. Approximately 0 % of women of child-bearing age carry GBS in the rectovaginal compartments (Park et al., 00). At birth, about 50 % of infants born to colonized mothers will also become colonized on their mucosal surfaces and on skin (Berner et al., 00). The mechanism of transmission from mother to offspring is still not very clear. A study by Berner et al. (00) hypothesized that the transmission of certain strains from mother to infant depends on a genetic polymorphism in the bacterial cell wall anchoring domain of the C protein â-antigen with its ability to bind human IgA on the mucosal surface. GBS are traditionally serotyped based on their capsular polysaccharide and protein antigens. Serotype V has been reported to be the most common serotype recovered from non-pregnant adults with invasive GBS disease (Blumberg et al., 996) and serotype III has been reported to be the most common serotype in neonatal invasive disease, accounting for 0 50 % of all cases (Berner et al., 00). Although serotyping is of great importance in epidemiological studies of GBS disease, it is not very discriminative and requires Abbreviations: GBS, group B streptococci; PFP, PFGE profile. specific reagents that may not be available in every laboratory. Hence molecular approaches which are reproducible and highly discriminatory for differentiating individual strains of bacterial pathogens are often applied to complement serotyping. These approaches include multilocus enzyme analysis (Quentin et al., 995), ribotyping (Huet et al., 99; Chatellier et al., 996), random amplified polymorphic DNA analysis (Chatellier et al., 997; Limansky et al., 998; Martinez et al., 000) and PFGE (Fasola et al., 99; Gordillo et al., 99; Rolland et al., 999; Le Thomas-Bories et al., 00; Benson et al., 00; Moyo et al., 00), analysis of insertion sequences (Rolland et al., 999; Dmitriev et al., 004), analysis of mobile genetic elements (Kong et al., 00) and, more recently, multilocus sequence typing (Jones et al., 00). Of these, PFGE is the most versatile and powerful technique for the study of chromosomal relatedness among bacterial strains as it is reproducible and very discriminative. Many researchers have demonstrated the discriminatory ability and reliability of PFGE in further differentiation of GBS strains of the same serotype (Rolland et al., 999; Le Thomas-Bories et al., 00; Benson et al., 00; Moyo et al., 00). In this study, we examined a collection of clinical strains of GBS by PFGE with the objective of assessing the genetic diversity of these strains, to determine the clonal relationship of GBS strains isolated from mothers and their infants and also the genetic relatedness of the strains obtained from different body sites from GBS-positive women. To the best of our knowledge, this is the first report on the molecular 0584 & 004 SGM Printed in Great Britain IP: On: Wed, 6 Dec 08 0:8:6

2 K.-L. Thong and others characterization of GBS from women and neonates in Malaysia. METHODS Bacterial strains. Over months in 00, all women admitted to the labour ward of the University Hospital, Kuala Lumpur, and their newborns subsequently delivered were sampled for GBS carriage. Vaginal, urethral and rectal swabs were taken from the women in labour, after obtaining informed consent. The specimens were placed in transport medium for transportation to the laboratory on the day of collection. External ear, mouth and umbilical swabs were taken from newborns after bathing but before transfer to the newborn nursery. Swabs were incubated overnight in Todd Hewitt broth (Oxoid) containing nalidixic acid and subcultured onto blood agar plates for the isolation of GBS. Isolates were identified by colonial morphology, Gram stain reaction, the CAMP test (Christie et al., 994) and Streptex latex agglutination (Murex Biotech). Isolates were kept at 80 8C for up to 9 months until used for PFGE analysis. DNA preparation and PFGE. Chromosomal DNA from each strain was prepared by the modified method as previously described (Thong et al., 00). Slices of DNA-containing agarose plugs were digested overnight with 0 units SmaI (Promega) at 5 8C, then electrophoresed on a CHEF DRII/III system (Bio-Rad Laboratories) for 4 h at 6Vcm, with ramped pulsed times of 40 s in a. 0 % (w/v) 0. 5 TBE gel. A lambda DNA concatemer PFGE marker (New England BioLabs) was used as a DNA size standard. The gel was stained with ethidium bromide ( ìgml ; Sigma) for 5 min, destained in distilled water for 5 min, and photographed under UV illumination. Data analysis. DNA fragment patterns were assessed visually, and distinct profiles were assigned an arbitrary PFGE profile (PFP). Dice coefficients of similarity (F) were calculated to compare the macrorestriction patterns. This coefficient, F, expresses the proportion of shared DNA fragments in two isolates and was calculated by the formula F ¼ n XY /(n X +n Y ), where n X is the total number of DNA fragments from isolate X, n Y is the total number of DNA fragments from isolate Y, and n XY is the number of DNA fragments that were identical in the two isolates. By this assessment F ¼. 0 indicates complete identity and F ¼ 0 indicates complete dissimilarity. Clustering was based on the unweighted pair group average method (UPGMA) and was performed with GelCompar II version.0 (Applied Maths, Kortrijk, Belgium). RESULTS PFGE analysis of SmaI-digested chromosomal DNA of GBS strains gave reproducible and discernible profiles. Among the strains studied, 48 distinct PFPs were identified. The PFPs consisted of 8 9 DNA fragments each, with F values of , indicating wide genetic variation in these strains. Representative SmaI profiles are shown in Fig.. A schematic representation of all the PFPs and the deduced genetic relationship of 48 PFPs are depicted in Fig.. Five distinctive clusters were identified, with each cluster consisting of different yet closely related PFPs. The most common profile obtained was S, as seen in 7. 4 % of all strains studied. Distribution of PFPs in relation to the source of GBS strains Mother infant pairs. Twelve sets of paired mother infant strains were analysed. Among these, 0 different SmaI kb Fig.. Representative SmaI PFGE profiles of Malaysian GBS strains. Lanes,,, and 9, lambda DNA concatemer PFGE marker; lanes and 5, DNA degraded; lanes 4, 6-7, 0, -, 6, 9-0, 4-5, SmaI PFGE profile S from isolates GBS 5, 7-8, -4, 6, 44-45, 50-5; lane, profile S (GBS ); lane 5, profile S4 (GBS 5); lanes 8-9, profile S5 (GBS 9-0); lane 4, profile S6 (GBS 5); lanes 5 and 0, profile S9 (GBS 5); lanes 6 and, profile S7 (GBS 6 and 6); lanes 7-8, profile S (GBS 9-0); lane 9, profile S8 (GBS ); lane, profile S0 (GBS 7); lane 4, profile S4 (GBS 0); lane 7, profile S6 (GBS 8); lane 8, profile S8 (GBS 40); lane, profile S0 (GBS 47); lane, profile S (GBS 48); lane 6, profile S (GBS 5); lane 7, profile S4 (GBS 59); lane 8, profile S5 (GBS 6); lane 40, profile S46 (GBS ). 99 IP: Journal of Medical Microbiology 5 On: Wed, 6 Dec 08 0:8:6

3 PFGE analysis of Streptococcus group B PFGE PFPs No of isolates S S S S0 S 4 S4 S S9 S5 S4 S45 S46 S5 S47 S40 S4 S48 S8 S8 S5 S9 S S8 S9 S7 S44 S6 S S4 S0 S4 S 5 S7 S6 S 47 S4 S S6 S7 S8 S6 S7 S0 4 S S9 S S5 S4 Fig.. Dendrogram showing the cluster analysis of the different SmaI PFGE profiles from clinical isolates of GBS strains, generated by the GelCompar program using the UPGMA method, based on the matrix of F values. IP: On: Wed, 6 Dec 08 0:8:6

4 K.-L. Thong and others Table. SmaI PFGE profiles of mother infant GBS pairs All the isolates were recovered from high vaginal swabs. No. Infant/mother Strain no. PFGE profile Infant GBS S Mother GBS S Infant GBS S Mother GBS 4 S Infant GBS 5 S Mother GBS 6 S4 4 Infant GBS 7 S Mother GBS 8 S 5 Infant GBS 9 S5 Mother GBS 0 S5 6 Infant GBS S Mother GBS S 7 Infant GBS S Mother GBS 4 S 8 Infant GBS 7 S Mother GBS 8 S 9 Infant GBS 9 S Mother GBS 0 S 0 Infant GBS S8 Mother GBS S8 Infant GBS S0 Mother GBS 4 S0 Infant GBS 5 S6 Mother GBS 6 S7 Table. SmaI PFGE profiles of isolates from different body sites within an individual (GBS-positive mother) Patient no. Strain no. Source PFGE profile GBS 44 Vaginal S GBS 45 Urethral S GBS 46 Vaginal S0 GBS 47 Urethral S0 GBS 48 Vaginal S GBS 49 Urethral S 4 GBS 50 Vaginal S GBS 5 Urethral S 5 GBS 55 Vaginal S GBS 5 Urethral S 6 GBS 54 Vaginal S GBS 5 Urethral S 7 GBS 56 Vaginal S GBS 57 Urethral S 8 GBS 58 Vaginal S4 GBS 59 Urethral S4 9 GBS 60 Vaginal S5 GBS 6 Urethral S5 0 GBS Vaginal S GBS 7 Rectal S6 GBS 6 Vaginal S7 GBS 5 Urethral S9 GBS 6 Rectal S7 profiles were seen (Table ; Fig., lanes 6 and 7, 8 and 9, and ). Only two mother infant pairs showed dissimilar profiles (S and S4 and S6 and S7). Most of the neonatal isolates were clustered in one group (Fig. ). The indistinguishable profiles between mother and infant in each pair reinforced earlier belief that most newborns acquire GBS from contamination by the maternal vaginal flora during the birth process. kb Genetic diversity of strains from different body sites within an individual. Twenty-three specimens ( vaginal, 0 urethral and rectal) from women were analysed to determine the genetic variability of strains isolated from different body sites within an individual (Table ). In nine women who gave both vaginal and urethral swabs, the vaginal isolates were indistinguishable from the urethral isolates, indicating autoinoculation as the likely mechanism of spread in the individual colonized at more than one body site (Fig. ). In the tenth woman, the vaginal isolate differed from the rectal isolate by five bands, and in the eleventh woman from whom three isolates were obtained from the vagina, urethra and rectum separately, the vaginal and rectal isolates were indistinguishable while the urethral isolate was different (Table ). These results suggest that some women may be colonized by multiple clones of GBS. GBS from neonates of culture-negative mothers. GBS was isolated from 7 neonates whose mothers were culturenegative for that organism (Table ). These 7 isolates yielded 9 different PFPs with S as the most common profile (47. %). In these infants, the possibility of an environmental 994 IP: Journal of Medical Microbiology 5 On: Wed, 6 Dec 08 0:8: Fig.. Representative SmaI PFGE profiles of paired GBS strains recovered from different sites from individuals. Lanes:, lambda DNA concatemer PFGE marker; and, paired vaginal urethral strains from patient 7 (S); 4 and 5, paired vaginal urethral strains from patient 9 (S5); 6 and 7, paired vaginal urethral strains from patient 6 (S); 8 and 9, paired vaginal urethral strains from patient 5 (S).

5 PFGE analysis of Streptococcus group B Table. SmaI PFGE profiles of GBS isolates from neonates of culture-negative mothers Table 4. SmaI PFGE profiles of strains from pregnant women with culture-negative infants No. Strain no. PFGE profile GBS 8 S GBS 9 S GBS 0 S4 4 GBS S9 5 GBS S5 6 GBS S 7 GBS 4 S 8 GBS 5 S 9 GBS 6 S 0 GBS 7 S GBS 8 S6 GBS 9 S7 GBS 40 S8 4 GBS 4 S 5 GBS 4 S9 6 GBS 4 S 7 GBS S source for their GBS cannot be excluded although it is also likely that the negative maternal cultures were due to technical errors. Genetic diversity of strains from pregnant women. From 59 clinical specimens taken from pregnant women whose newborn infants were culture-negative for GBS, 0 profiles were obtained (F ¼ ) with profile S again the most common PFP (4 %) (Table 4). Except for PFPs S0 and S0 S4 (which were also seen in other individuals Table ), most of the profiles were unique and were represented by one isolate each. This shows the wide genetic diversity of Malaysian GBS strains. DISCUSSION Molecular typing of GBS has distinct advantages over the traditional serotyping used to identify sources of infection in early and late-onset neonatal disease and for the investigation of nosocomial outbreaks. Restriction endonuclease analysis of chromosomal DNA (REA) and ribotyping have been shown to be reliable in the demonstration of epidemiological relationships and are able to differentiate among strains of the same serotypes (Blumberg et al., 99). Harrison et al. (995) used REA to distinguish relapse from reinfection in recurrent GBS disease. Limansky et al. (998) also showed that GBS reactivation and reinfection could be differentiated by RAPD. Gordillo et al. (99) found that PFGE patterns obtained with SmaI digestion of GBS strains were more easily discerned and interpreted than patterns generated by conventional electrophoresis with HindIII or EcoRI plus BglII. Similarly, Fasola et al. (99) and Ferrieri et al. (997) obtained more SmaI PFGE patterns than REA with HindIII or HindIII followed by EcoRI and conventional agarose gel SmaI profile Source (no. of strains) SmaI profile Source (no. of strains) S Vaginal (0) S4 Vaginal () Urethral (4) S5 Vaginal () S0 Urethral () S6 Vaginal () S Vaginal () S7 Vaginal () Rectal () S Vaginal () S8 Vaginal () S Vaginal () S9 Vaginal () S4 Vaginal () S40 Vaginal () S5 Vaginal () S4 Vaginal () S6 Vaginal () S4 Vaginal () Urethral () S7 Vaginal () S4 Vaginal () S8 Vaginal () S44 Vaginal () Urethral () S9 Vaginal () S45 Vaginal () S0 Vaginal () S46 Vaginal () S Vaginal () S47 Vaginal () S Urethral () S48 Vaginal () S Vaginal () electrophoresis. Many researchers who used PFGE analysis of GBS strains have documented considerable genetic diversity among strains of similar serotypes (Fasola et al., 99; Rolland et al., 999; Le Thomas-Bories et al., 00; Moyo et al., 00). In this study, we have examined GBS strains isolated from mothers and newborn infants by using PFGE analysis with SmaI digestion. The results illustrated the wide genetic diversity in strains of GBS in the female genital tract, mother infant transmission during the birth process, the co-existence of different clones of GBS in the same individual and the clonal spread of strains to different body sites in the same individual. At our hospital, GBS from the urogenital tract had previously been studied by serotyping with the capillary precipitin test, and serotype was shown to be the predominant type (50. 9 %) among both infants and adults, with identical serotypes found in mother and infant pairs (Ngeow & Puthucheary, 987). In the present study on carrier strains, there was also a predominant profile (S) occurring in 46/ (7. 4 %) strains. Although pathogenic strains of GBS may be genetically different from carrier strains, it is expected that PFGE patterns of isolates from GBS-infected patients would similarly show a much wider genetic diversity than what was previously determined by serotyping. The higher discriminatory power of PFGE analysis identified a few non-identical PFGE patterns among mother infant pairs, thus raising the possibility of environmental contamination of the infant by GBS occurring soon after birth. The discrepancy in PFGE patterns found in mother infant pairs IP: On: Wed, 6 Dec 08 0:8:6

6 K.-L. Thong and others can also be the result of transmission of only one of several clones of GBS colonizing the mother or, more likely, the laboratory selection (from each culture examined) of only one of several clones of GBS present in both mother and infant. These possibilities can be further examined by the selection of more colonies from both maternal and neonatal cultures for genotyping. Overall, our data confirmed the other researchers reports of the heterogeneous nature of individual GBS serotypes by PFGE analysis (Gordillo et al., 99; Ellis et al., 996). Overall, the Malaysian strains of GBS were defined by 48 profiles. Thirty PFPs were unique, with each represented by a single isolate. This demonstrates the high discriminatory ability of PFGE in differentiating very closely related strains. The existence of different PFGE types in the same individual may be the result of mutation or co-colonization with strains from different sources, e.g. from different sex partners. This concurrent carriage of different genotypes has to be borne in mind when genotypic information is used to establish proof of epidemiological linkage in disease transmission. It may be necessary for the laboratory to test several isolates from a carrier or case before the individual can be excluded as the source of an outbreak of infection on the basis of a difference in genotype pattern. The changing epidemiology of GBS colonization reported in recent years (Hickman et al., 00) underlines the importance of continued GBS surveillance for the design of GBS vaccines. The PFGE analysis we used fulfilled the criteria of a useful typing method in that it is reproducible, has sufficient discriminatory power, is able to type all strains and is practical for the hospital laboratory that has to carry out nosocomial infection investigations. Compared to the newer approach of multilocus sequencing typing (Jones et al., 00), PFGE is relatively cheaper and more accessible to most laboratories. It should also be an appropriate typing method for the study of the association of genetic types with disease manifestation, development and response to treatment. ACKNOWLEDGEMENTS The authors would like to thank Drs Tetty Aman Nasution and Yi Yi for technical assistance. Funding for the study comes from the University of Malaya F-Vote and IRPA grant , and from the Ministry of Science, Technology and Environment, Malaysia. REFERENCES Benson, R. D., Luchansky, J. B., Eliot, J. A., Dagnan, A. J., Willenberg, H. J., Thonberg, J. M. & Kaya, H. H. (00). Pulsed field fingerprinting of vaginal Group B Streptococcus in pregnancy. Obstet Gynecol 00, Berner, R., Csorba, J. & Brandis, M. (00). Different cytokine expression in cordblood mononuclear cells (CBMNC) after stimulation with neonatal sepsis or colonizing strains of Streptococcus agalactiae. Pediatr Res 49, Berner, R., Ruess, M., Bereswill, S. & Brandis, M. (00). Polymorphisms in the cell wall-spanning domain of the C protein â-antigen in clinical Streptococcus agalactiae isolates are caused by genetic instability of repeating DNA sequences. Pediatr Res 5, 06. Blumberg, H. M., Stephens, D. S., Licitra, C., Pigott, N., Facklam, R., Swaminathan, B. & Wachsmuth, I. K. (99). Molecular epidemiology of group B streptococcal infections: use of restriction endonuclease analysis of chromosomal DNA and DNA restriction fragment length polymorphisms of ribosomal RNA genes (ribotyping). J Infect Dis 66, Blumberg, H. M., Stephens, D. S., Modansky, M. & 6 other authors (996). Invasive group B streptococcal disease: the emergence of serotype V. J Infect Dis 7, Chatellier, S., Huet, H., Kenzl, S., Rosenau, A., Geslin, P. & Quentin, R. (996). Genetic diversity of rrna operons of unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid of neonates suffering from meningitis. J Clin Microbiol 4, Chatellier, S., Ramanatson, C., Harriau, P., Rolland, K., Roseanau, A. & Quentin, R. (997). Characterization of Streptococcus agalactiae strains by randomly applied polymorphic DNA analysis. J Clin Microbiol, Christie, R., Atkins, N. E. & Munch-Peterson, E. (994). A note on lytic phenomenon shown by group B streptococci. Aust J Exp Biol Med Sci, Dmitriev, A., Shen, A., Shen, X. & Yang, Y. (004). ISSa4-based differentiation of Streptococcus agalaticae strains and identification of multiple targets for ISSa4 insertions. J Bacteriol 86, Ellis, S., Kotina, M. & Garland, S. M. (996). Restriction endonuclease analysis of group B streptococcal isolates from two distinct geographical regions. J Hosp Infect, Fasola, E., Livdahl, C. & Ferrieri, P. (99). Molecular analysis of multiple isolates of the major serotypes of group B streptococci. J Clin Microbiol, Ferrieri, P., Cho, N. S., Livdahl, C., Rubens, C. E. & Flores, A. E. (997). DNA restriction profiles of nontypable group B streptococcal clinical isolates. Adv Exp Med Biol 48, Gordillo, M. E., Singh, K. V., Baker, C. J. & Murray, B. E. (99). Typing of group B streptococci: comparison of pulsed-field gel electrophoresis and conventional electrophoresis. J Clin Microbiol, Harrison, L. H., Ali, A., Dwyer, D. M. & 6 other authors (995). Relapsing invasive group B streptococcal infection in adults. Ann Intern Med, Hickman, M. E., Rench, M. A., Ferrieri, P. & Baker, C. J. (00). Changing epidemiology of group B streptococcal colonization. Pediatrics 04, Huet, H., Martin, C., Geslin, P., Grimont, F. & Quentin, R. (99). Ribotyping of Streptococcus agalactiae strains isolated from vaginas of asymptomatic women. Res Microbiol 44, Jones, N., Bohnsack, J. F., Takashahi, S. & 9 other authors (00). Multilocus sequence typing system for group B Streptococcus. J Clin Microbiol 4, Kong, F., Martin, D., James, G. & Gilbert, G. L. (00). Towards a genotyping system for Streptococcus agalactiae (group B streptococcus): use of mobile genetic elements in Australasian invasive isolates. J Med Microbiol 54, Le Thomas-Bories, I., Fitoussi, F., Mariani-Kurkdjian, P., Raymond, J., Brahimi, N., Bidet, P., Lefrance, V. & Bingen, E. (00). Clonal relationship between USA and French Serotype V group B Streptococcus isolates. J Clin Microbiol 9, Limansky, A. S., Sutich, E. G., Guardati, M. C., Toresani, I. E. & Viale, A. M. (998). Genomic diversity among Streptococcus agalactiae isolates detected by a degenerate oligonucleotide primed amplification array. J Infect Dis 77, IP: Journal of Medical Microbiology 5 On: Wed, 6 Dec 08 0:8:6

7 PFGE analysis of Streptococcus group B Martinez, G., Harel, J., Higgins, R., Lacouture, S., Daignault, D. & Gottschalk, M. (000). Characterization of Streptococcus agalactiae isolates of bovine and human origin by randomly amplified polymorphic DNA analysis. J Clin Microbiol 8, Moyo, S. R., Maeland, J. A. & Bergh, K. (00). Typing of human isolates of Streptococcus agalactiae (group B Streptococcus, GBS) strains from Zimbabwe. J Med Microbiol 5, Ngeow, Y. F. & Puthucheary, S. D. (987). Distribution of group B streptococcal serotypes in Malaysia. J Trop Med Hyg 90, Park, C. J., Vandel, N. M., Ruprai, D. K., Martin, E. A., Gates, K. M. & Coker, D. (00). Detection of group B streptococcal colonization in pregnant women using direct latex agglutination testing of selective broth. J Clin Microbiol 9, Quentin, R., Huet, H., Wang, F. S., Geslin, P., Goudeau, A. & Selander, R. K. (995). Characterization of Streptococcus agalactiae strains by multilocus enzyme genotype and serotype: identification of multiple virulent clone families that cause invasive neonatal disease. J Clin Microbiol, Rolland, K., Marois, C., Siquier, V., Cattier, B. & Quentin, R. (999). Genetic features of Streptococcus agalactiae strains causing severe neonatal infections, as revealed by pulsed field gel electrophoresis and hylb gene analysis. J Clin Microbiol 7, Thong, K. L., Nasir, M. D., Goh, Y. L., Ngeow, Y. F. & Parasakthi, N. (00). Rapid pulsed-field gel electrophoresis method for Streptococcus spp. Asia Pac J Mol Biol Biotechnol 9, IP: On: Wed, 6 Dec 08 0:8:6