Quantitative analysis of the e ect of xylitol on pneumococcal nasal colonisation in rats

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1 FEMS Microbiology Letters 178 (1999) 313^317 Quantitative analysis of the e ect of xylitol on pneumococcal nasal colonisation in rats Tero Kontiokari a; *, Martti Svanberg b, Pauli Mattila b, Maija Leinonen c, Matti Uhari a Abstract c a Department of Paediatrics, University of Oulu, FIN Oulu, Finland b Department of Dentistry, University of Oulu, FIN Oulu, Finland National Public Health Institute, Department in Oulu, FIN Oulu, Finland Received 4 June 1999; received in revised form 22 July 1999; accepted 23 July 1999 Xylitol possesses anti-bacterial effects on pneumococci in vitro. To study the effect in vivo, the nostrils of 80 rats were inoculated with pneumococci. Intervention groups (n = 20) received either a xylitol diet or xylitol nasal sprays. The control groups were on a normal diet or had saline sprays. After 3 days, a quantitative bacterial culture and a PCR were done from the mucosal suspension. Neither the mean colony-forming unit counts nor the PCR counts differed statistically significant between the xylitol and control groups. Thus, we found that xylitol had no significant effect on pneumococcal mucosal colonisation. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Xylitol; Sugar alcohol; Colonisation; Anti-microbial; Prevention; Rat 1. Introduction Xylitol is a food additive which has anti-bacterial properties against K-haemolytic streptococci, including Streptococcus pneumoniae [1^4]. Acute otitis media (AOM) can be prevented by orally administered xylitol [5,6]. A local mucosal anti-microbial e ect has been proposed to be the mechanism of action, but orally given xylitol has had no e ect on the nasopharyngeal carriage of pneumococci in children [5]. A similar lack of correlation between pneumococcal carriage and sul soxazole in view of the preventive e cacy on clinical disease has also been observed [7]. This may be due to the fact that nasopharyngeal swab sampling is di cult to standardise and that these samples do not necessarily represent the true carriage status. Because of these limitations implicit in clinical trials, we decided to undertake a study to evaluate whether xylitol has an e ect on the quantity of bacteria in a rat model of nasal carriage of pneumococci. We also evaluated whether nasal administration of xylitol provides some advantages compared to the oral route. 2. Materials and methods * Corresponding author. Tel.: +358 (8) ; Fax: +358 (8) ; tero.kontiokari@oulu. We used 80 3-month old Sprague-Dawley rats / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (99)

2 314 T. Kontiokari et al. / FEMS Microbiology Letters 178 (1999) 313^317 (Laboratory Animal Centre, University of Oulu, Oulu, Finland), 20 in each intervention group, which were kept in separate cages. The Ethical Committee on Animal Experiments of the University of Oulu approved the study protocol. One group (controls) was fed a basal powder diet, RM1 (Special Diet Services, Witham, Essex, UK), throughout the study. Another group got food supplemented with xylitol (Xyro n, Kotka, Finland) in such a way that 20% of the total dry weight of the daily food consisted of xylitol. The diet was started 1 week before the bacterial challenge in order to make the rats adapted to xylitol. The two other groups got nasal sprays ve times per day, 100 Wl in each nostril, starting 24 h before the inoculation of bacteria, and this treatment was continued during the whole experiment. One group (controls) got saline sprays and the other isotonic xylitol sprays. The xylitol suspension was prepared by adding 5% (w/v) xylitol to distilled aqua (Pharmacy of the University Hospital of Oulu, Oulu, Finland). The pneumococcal strains used were from patients` middle ear aspirates and they were serotyped as described earlier [8] and kept frozen in skim milk at 320³C until used. After that, they were grown in Brain Heart Infusion medium (Difco Laboratories, Detroit, MI, USA) to the exponential phase [3] and harvested by centrifugation for 15 min at 2500Ug. The precipitate was suspended in phosphate bu er at ph 7.4 and the colony-forming units (cfu) of the suspension were measured with the standard plate dilution method on a blood agar plate. The bacterial inoculation was done in two separate stages as illustrated in Fig. 1. In the rst experiment, each rat received a total of 3U10 4 cfu of serotype 19F, while the rats in the second experiment got a mixture containing 2^4U10 6 cfu of each of the three serotypes, 6B, 19F and 23F. These inoculation doses were chosen to induce carriage without causing septichemia [9]. The rats were decapitated at day 3. The nasoturbinates and nasal mucosa were removed sterilely. The samples were weighed and diluted to 1 ml of phosphate bu er at ph 7.4. The samples were then sterilely homogenised with an Ultra Turrex 1020 tissue grinder (Ystral, Dottingen, Germany) and cultured on gentamicin blood agar plates. The culturing was done in dilutions of 10, starting with 10 Wl of the original solution and using a detection level of 10 2 cfu per sample. Each experiment was done at least in duplicate. The cfu were counted at 24 and 48 h and the bacteria were serotyped to be one of the inoculated strains from at least three di erent colonies. The mean of the last two positive dilutions was counted and the cfu count was then given as a mean of re- Fig. 1. The inoculation of bacteria to rat nasal mucosa. First (1), inoculation was done with one serotype (19F) given to 10 rats in each group and second (2), with a mixture of three serotypes (6B, 19F and 23F) given to another 10 rats in each group. In the rst experiment, the bacteria were used in a concentration of 1.5U10 6 cfu ml 31 and in the second experiment, in concentrations of 4, 3 and 6U10 8 cfu ml 31 of the serotypes 6B, 19F and 23F, respectively. 10 Wl of the suspension was inoculated to both nostrils of the rat. Thus, each rat in the rst experiment received a total of 3U10 4 bacteria of serotype 19F, while the rats in the second experiment got 2^4U10 6 bacteria in a mixture containing each of the three serotypes, 6B, 19F and 23F.

3 T. Kontiokari et al. / FEMS Microbiology Letters 178 (1999) 313^ Table 1 The presence of S. pneumoniae in the nasal mucosa of rats demonstrated by a quantitative bacterial culture and by semi-quantitative PCR in four intervention groups (n = 20 in each group) peated experiments. Pneumococcal DNA was demonstrated from the original suspension by using a PCR technique described earlier [10]. Statistical analyses were performed separately on the diet groups and the nasal spray groups. The differences in the proportions of positive and negative cultures and PCR were compared with the chisquare test and the di erences of the means of cfu were tested with the t-test. 95% Con dence intervals for the di erences were calculated. Before the trial, a preliminary experiment with nine rats was performed. It showed that pneumococcal colonisation can be induced with the method described above and no pneumococci were found before the inoculation. 3. Results Diet Pneumococcal culture was positive in eight (40%), 12 (60%), 13 (65%) and 12 (60%) samples in the control, xylitol, saline spray and xylitol spray groups, respectively, and none of the di erences were statistically signi cant (Table 1). The corresponding mean bacterial counts were 24, 33, 115 and 68 cfu g 31, respectively, none of the di erences being statistically signi cant (Table 1). Pneumococcal DNA was present in nine (45%) and 11 (55%) cases in the control and xylitol diet groups, respectively, while the corresponding number in the two spray groups was 14 (70%). The di erences were not statistically signi cant (Table 1). All animals were free of symptoms before decapitation. The high nasal challenge dose (10 6 ) of bacteria resulted in a positive culture on day 3 more often than the low dose (10 4 ) (28/40 versus 17/40 positive cultures, di erence 28%, con dence interval (CI) 6^ 47, P = 0.013). The serotypes 19F and 23F, but not serotype 6B, could be demonstrated in these cultures. The serotypes were distributed equally between the intervention groups (Table 2). 4. Discussion Nasal spray Control Xylitol Di erence (CI) P Saline 5% xylitol Di erence (CI) P Positive culture (%) 8 (40) 12 (60) 320% (348 to 11%) 0.12 a 13 (65) 12 (60) 5% (325 to 34%) 0.74 a Positive PCR (%) 9 (45) 11 (55) 310% (339 to 21%) 0.53 a 14 (70) 14 (70) 0% (328 to 28%) 1.0 a Mean growth (S.D.) U10 4 cfu g (36) 33 (63) 39 (342 to 24) 0.58 b 115 (109) 68 (97) 47 (319 to 113) 0.16 b CIs (95%) have been calculated for the di erences. The di erences between the diet groups and between the spray groups were not statistically signi cant. a Chi-square test. b t-test. Rodent models have been recommended for the use in comparative studies evaluating the e ect of various interventions on mucosal bacterial colonisation [9,11]. They have good reproducibility and mucosal cultures represent reliably stable colonisation Table 2 Number of animals colonised by pneumococci according to the di erent serotypes of S. pneumoniae in the intervention groups Intervention (n = 20 in each group) Serotype Control diet Xylitol diet Saline spray 5% Xylitol spray 19F F Both 19F+23F Serotyping was done from at least three di erent colonies.

4 316 T. Kontiokari et al. / FEMS Microbiology Letters 178 (1999) 313^317 rather than only the temporal surface contamination by the inoculum [9,11]. In accordance with the earlier observations, we found colonisation to be dependent on the bacterial dose. The mean bacterial count in nasal mucosa showed large variation from 24 to 115U10 4 cfu g 31 in the control groups. This is in contrast to what was observed in a mouse model with super cial rinsing sampling, where the number of cfu carried has been rather stable and generally between 10 3 and 10 4 cfu [9]. The precise mechanism of nasopharyngeal pneumococcal colonisation is unclear. The dose and the phase of growth of the invading bacteria as well as the adhesive properties of both the bacteria and the nasopharyngeal cells have been found to be important [9,11,12]. As xylitol is able to decrease pneumococcal growth and adhesion in vitro, an in uence on pneumococcal carriage would be the most logical explanation for its ability to prevent AOM [3^6]. In contrast to this hypothesis, we found xylitol to be ine ective in preventing pneumococcal colonisation. The daily amount of xylitol in the diet group was at least 3-fold compared to the dose used in the clinical studies in which the protective e ect against AOM was observed [5,6]. Yet, no di erences in the mean cfu counts or the presence of pneumococcal DNA were found between the diet regimens. The nasal dosage maximised the local e ect. The volume of 2U100 Wl of xylitol suspension lled the whole nasal cavity and ensured a bacteriostatic e ect against pneumococci at least at the time of administration, but this dosage was not e ective in reducing the number of pneumococci signi cantly compared to saline. Thus, xylitol had neither a local nor a systemic e ect on pneumococcal carriage, irrespective of the xylitol dosage or the methods by which colonisation was evaluated. The small sample size and the detection level of 10 2 bacteria per sample ensure that only the major e ects of xylitol on the number of pneumococci in nasal mucosa are seen, but it seems unlikely that the decrease up to 40% in the AOM rate could be explained by any small decrease in carriage. Pneumococcus is the most common bacterium causing AOM and the serotypes selected to be used in this study represent the three serotypes most frequently isolated from middle ear e usion. There were no di erences in the serotype distribution of pneumococci between the groups either. Our results do not exclude the possible e ect of xylitol on bacterial growth during viral in ammation, when the bacterial load increases and new strains are acquired which are more capable of causing infection than the strains which have been present longer [13]. Nasopharyngeal pneumococcal colonisation is associated with AOM and bacterial carriage increases during viral in ammation as well as during AOM [13^16]. However, the studies are inconclusive as to whether the healthy-state carriage is a risk factor for AOM per se [13^18]. In conclusion, xylitol was ine ective in preventing the nasal colonisation of pneumococci in rats, irrespective of the mode of administration, and it had no selective e ect on the colonising strains. As this nding is in accordance with our earlier observation in children [5], it seems evident that an e ect on pneumococcal carriage is not the mechanism of action of xylitol against AOM. Acknowledgements This study was supported by grants from the Maud Kuistila Foundation, Yrjo«Jahnsson Foundation and the Scandinavian Society of Antimicrobial Chemotherapy. References [1] Knuuttila, M.L.E. and Ma«kinen, K.K. (1975) E ect of xylitol on the growth and metabolism of Streptococcus mutans. Caries Res. 9, 177^189. [2] Vadeboncoeur, C., Trahan, L., Mouton, C. and Mayrand, D. (1983) E ect of xylitol on the growth and glycolysis of acidogenic oral bacteria. J. Dent. Res. 62, 882^884. [3] Kontiokari, T., Uhari, M. and Koskela, M. (1995) E ect of xylitol on growth of nasopharyngeal bacteria in vitro. Antimicrob. Agents Chemother. 39, 1820^1823. [4] Kontiokari, T., Koskela, M. and Uhari, M. (1998) Antiadhesive e ect of xylitol on otopathogenic bacteria. J. Antimicrob. Chemother. 41, 563^565. [5] Uhari, M., Kontiokari, T., Koskela, M. and Niemela«, M. (1996) Xylitol chewing gum in prevention of acute otitis media: double blind randomised trial. Br. Med. J. 313, 1180^ [6] Uhari, M., Kontiokari, T. and Niemela«, M. (1998) A novel

5 T. Kontiokari et al. / FEMS Microbiology Letters 178 (1999) 313^ use of xylitol sugar in prevention of acute otitis media. Pediatrics 102, 879^884. [7] Perrin, J.M., Charney, E., MacWhinney, J.B., McInerny, T.K., Miller, R.L. and Nazarian, L.F. (1974) Sul soxazole as chemoprophylaxis for recurrent otitis media: a double blind crossover study in pediatric practise. New Engl. J. Med. 291, 664^667. [8] Sankilampi, U., Herva, E., Haikala, R., Liimatainen, O., Renkonen, O.-V. and Leinonen, M. (1997) Epidemiology of invasive Streptococcus pneumoniae infections in adults in Finland. Epidemiol. Infect. 118, 7^15. [9] Wu, H.-Y., Virolainen, A., Mathews, B., King, J., Russel, M.W. and Briles, D.E. (1997) Establishment of a Streptococcus pneumoniae nasopharyngeal colonisation model in adult mice. Microb. Pathog. 23, 127^137. [10] Virolainen, A., Salo, P., Jero, J., Karma, P., Eskola, J. and Leinonen, M. (1994) Comparison of PCR assay with bacterial culture for detecting Streptococcus pneumoniae in middle ear uid of children with acute otitis media. J. Clin. Microbiol. 32, 2667^2670. [11] Weiser, J.N., Austrian, R., Sreenivasan, P.K. and Masure, H.R. (1994) Phase variation in pneumococcal opacity: relationship between colonial morphology and nasopharyngeal colonisation. Infect. Immun. 62, 2582^2589. [12] Andersson, B., Eriksson, B., Falsen, E., Fogh, A., Hanson, L.A î., Nylën, O., Peterson, H. and Svanborg-Edën, C. (1981) Adhesion of Streptococcus pneumoniae to human pharyngeal epithelial cells in vitro: di erences in adhesive capacity among strains isolated from subjects with otitis media, septicemia, or meningitis or from healthy carriers. Infect. Immun. 32, 311^ 317. [13] Faden, H., Stanievich, J., Brodsky, L., Berstein, J. and Ogra, P.L. (1990) Changes in nasopharyngeal ora during otitis media of childhood. Pediatr. Infect. Dis. J. 9, 623^626. [14] Faden, H., Waz, M.J., Bernstein, J.M., Brodsky, L., Stanievich, J. and Ogra, P.L. (1991) Nasopharyngeal ora in the rst three years of life in normal and otitis-prone children. Ann. Otol. Rhinol. Laryngol. 100, 612^615. [15] Homoe, P., Prag, J., Farholt, S., Henrichsen, J., Horsleth, A., Killan, M. and Jensen, J.S. (1996) High rate of nasopharyngeal carriage of potential pathogens among children in Greenland: results of a clinical survey of middle-ear disease. Clin. Infect. Dis. 23, 1081^1090. [16] Faden, H., Du y, L., Wasielewski, R., Wolf, J., Krysto k, D. and Tung, Y. (1997) Relationship between nasopharyngeal colonisation and the development of otitis media in children. J. Infect. Dis. 175, 1440^1445. [17] Stenstrom, C. and Ingvarsson, L. (1997) Otitis-prone children and controls: a study of possible predisposing factors. 2. Physical ndings, frequency of illness, allergy, day care and parental smoking. Acta Otolaryngol. 117, 696^703. [18] Prellner, K., Christensen, P., Hovelius, B. and Rosen, C. (1984) Nasopharyngeal carriage in otitis-prone and non-otitis-prone in day-care centres. Acta Otolaryngol. 98, 343^350.