Nasopharyngeal Streptococcus pneumoniae carriage in Japanese children attending day-care centers

International Journal of Pediatric Otorhinolaryngology 75 (2011) 664–669

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Nasopharyngeal Streptococcus pneumoniae carriage in Japanese children attending day-care centers Koichi Hashida, Teruo Shiomori, Nobusuke Hohchi, Jun-ichi Ohkubo, Toyoaki Ohbuchi, Takanori Mori, Hideaki Suzuki * Department of Otorhinolaryngology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 28 December 2010 Accepted 2 February 2011 Available online 2 March 2011

Objectives: We conducted a prospective bacteriological survey to investigate antibiotic resistancerelated genetic characteristics and the turnover of nasopharyngeal Streptococcus pneumoniae carriage in healthy children in day-care centers (DCCs). Methods: A total of 363 nasopharyngeal mucus samples were collected from children aged 0 to 6 years attending two DCCs in the summer of 2004 (n = 181) and the following winter (n = 182). We obtained 157 S. pneumoniae isolates and analyzed them by antibiotic susceptibility testing, PCR assay for the penicillin-binding protein (PBP) genes and macrolide-resistance gene, and pulsed-field gel electrophoresis (PFGE). Results: The overall carriage rate was 43.3% (157/363). The percentages of penicillin-intermediately resistant S. pneumoniae (PISP) strains, penicillin-resistant S. pneumoniae (PRSP) strains, erythromycinintermediately resistant S. pneumoniae strains and erythromycin-resistant S. pneumoniae strains were 35.7% (56/157), 0.6% (1/157), 1.9% (3/157), and 69.4% (109/157), respectively. The percentages of S. pneumoniae strains with the pbp mutation(s) and mefA and/or ermB gene(s) were 92.4% (145/157) and 71.3% (112/157), respectively. Fifty strains with different PFGE patterns were obtained from among the 157 isolates. Thirteen strains were observed in both seasons, but only one of these strains was isolated from the same carrier. Twenty-one strains (42.0%) were isolated from two or more children, and 17 of these were each isolated from children attending the same DCC. Conclusions: These results indicate the spread of S. pneumoniae, particularly those with antibioticresistance genes, and the vigorous genetic turnover and substantial horizontal transmission of this pathogen in healthy children attending DCCs in Japan. ß 2011 Elsevier Ireland Ltd. All rights reserved.

Keywords: Streptococcus pneumoniae Nasopharyngeal carriage Day-care Antibiotic resistance

1. Introduction The nasopharyngeal flora is generally thought to be a major reservoir for bacterial pathogens of respiratory tract infections (RTIs) in children [1–3]. In recent years, the morbidity of RTIs caused by antibiotic-resistant pathogens has been increasing in children, creating a worldwide health problem. Streptococcus pneumoniae (S. pneumoniae) and Haemophilus influenzae are the major bacterial pathogens colonizing the nasopharynx, and often cause RTIs. Penicillin resistance of S. pneumoniae develops predominantly by the alterations of penicillin-binding proteins (PBPs), PBP1A, PBP2B and PBP2X, due to mutations in corresponding genes (pbp1a, pbp2b, and pbp2x), that reduce the affinity of PBPs for blactams [4–6]. In the meantime, macrolide resistance of S.

* Corresponding author. Tel.: +81 93 691 7448; fax: +81 93 601 7554. E-mail address: [email protected] (H. Suzuki). 0165-5876/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2011.02.005

pneumoniae occurs via two different mechanisms; that is, modification of the target site of bacterial ribosomes by methylation that prevents the binding of the antibiotic to the target site, and the activation of an efflux pump to eliminate the antibiotic from the cell. These two mechanisms are known to be mediated by the ermB and mefA genes, respectively [7–9]. There has been growing awareness of increasing morbidity from community-acquired RTIs, particularly those caused by drug-resistant bacteria. Day-care-center (DCC) attendance of children has been reported to be one of the risk factors for RTI including acute otitis media [10–12], and for the nasopharyngeal carriage of bacteria such as S. pneumoniae and H. influenzae [2,13,14]. However, the molecular epidemiology of S. pneumoniae in the upper respiratory tract in connection with DCC attendance is not fully understood [13–19]. The aim of this study was to investigate antibiotic-resistance-related genetic characteristics and the turnover of nasopharyngeal S. pneumoniae carriage in children attending DCCs.

K. Hashida et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 664–669

2. Materials and methods 2.1. Subjects Children in two DCCs (DCC-A and DCC-B) in a local city in Fukuoka Prefecture in Japan were enrolled. None had been vaccinated against S. pneumoniae. The survey was performed in July 2004 (summer) and in February 2005 (winter). The summer survey included 181 children, 94 boys and 87 girls, ranging in age from 1 to 6 years with an average age of 3.0 years. The winter survey included 182 children, 95 boys and 87 girls, ranging in age from 0 to 6 years with an average age of 3.7 years. One hundred and forty-seven children underwent both surveys. Written questionnaires about present illness (otitis media, rhinosinusitis, tonsillitis, bronchitis, common cold, and allergic rhinitis) were completed by the children’s parents a few days before bacterial sampling. Informed consent was obtained from the parents, and the study was approved by the Ethics Committee of the University of Occupational and Environmental Health. 2.2. Bacterial sampling and culture Nasopharyngeal mucus was transnasally collected with a sterile cotton swab (Seed Swab No. 2; Eiken Chemical Co., Ltd., Tokyo, Japan) and subjected to bacterial culture. The samples were placed on 5% sheep blood agar plates (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), and incubated under a humidified atmosphere containing 5% CO2 at 35 8C overnight. S. pneumoniae was identified by standard microbiological procedures as previously described [20]. The isolates were stored in a stock solution (Microbank; ProLab Diagnostics, Ontario, Canada) at 80 8C until the following analyses. 2.3. Antimicrobial susceptibility The penicillin minimum inhibitory concentrations (MICs) were determined by the broth microdilution method, in accordance with the guidelines of the Japanese Society of Chemotherapy [21], using an Eiken Dry Plate (cation-adjusted Mueller– Hinton broth with NAD (15 mg/l), yeast extract (5 g/l), and lysed horse blood (20 ml/l)) [22]. Briefly, each well of the plate was inoculated with a 100-ml inoculum containing approximately 5  105 CFU, and the plate was incubated at 35 8C for 20 h. The MIC was read as the lowest concentration of an antimicrobial agent at which there was no visible growth. Breakpoints for susceptibility were provided according to the guidelines of the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) [23]. Isolates with MICs for penicillin of 0.06 mg/ml, 0.12–1 mg/ml, and 2 mg/ml were judged to be penicillin susceptible, intermediately resistant, and resistant, respectively. Those with MICs for erythromycin of 0.25 mg/ml, 0.5 mg/ml, and 1 mg/ml were judged to be erythromycin susceptible, intermediately resistant, and resistant, respectively. 2.4. PCR analysis Six sets of primers (Wakunaga Pharmaceutical Co., Ltd., Hiroshima, Japan) were used [24–26]: primers for detecting the lytA gene encoding the autolysin enzyme specific to S. pneumoniae, the pbp1a gene, the pbp2x gene, the pbp2b gene, the mefA gene, and the ermB gene. The primers for the three PBP genes were designed to amplify certain segments of the normal pbp1a, pbp2x, and pbp2b genes, which are present only in penicillin-susceptible strains [24]. The mefA and ermB genes are responsible for resistance to macrolides [24].

665

A single colony of S. pneumoniae on the sheep blood agar plate was collected and suspended in 30 ml of lysis solution (3 ml of 1 M Tris–HCl (pH 8.9), 6 mg of proteinase K, 0.225% Tween 20, 0.225% Nonidet P-40, 3 ml of 10 Tth polymerase buffer) [27], and heated at 60 8C for 10 min and at 94 8C for 5 min in a thermal cycler (TaKaRa PCR Thermal Cycler MP; Takara Bio Co., Ltd., Shiga, Japan). Two ml of the bacterial lysate was then dispensed to each of three tubes, which contained two sets of primers for the lytA and pbp1a genes, for the pbp2x and the pbp2b genes, and for the mefA and the ermB genes, respectively. The reaction mixture consisted of 3 ml of 10 PCR buffer, 3 ml of a deoxynucleoside triphosphate mixture at a concentration of 2 mM, 1.2 U of Tth DNA polymerase (Toyobo Co., Ltd., Osaka, Japan), and 4 pmol of both sense and reverse primers in a total volume of 30 ml. The PCR cycling conditions consisted of 30 cycles of 94 8C for 15 s, 53 8C for 15 s, and 72 8C for 15 s. Amplified DNA fragments were analyzed by gel electrophoresis using 3% agarose. 2.5. Pulsed-field gel electrophoresis (PFGE) PFGE analysis was carried out by a modification of the method of Yano et al. [18]. The bacteria were suspended in 50 mM EDTA (pH 8.0) at a concentration of 1 McFarland. The pellet was mixed with an equal volume of melted 1.2% chromosomal-grade agarose (Bio-Rad Laboratories, Richmond, CA, USA). The mixture was chilled in an insert former, and the resultant plugs were treated with 0.5 ml of lysis solution I (6 mM Tris–HCl (pH 8.0), 0.1 M EDTA, 0.5% Brij 58, 0.2% sodium deoxycholate, 0.5% Sarkosyl, 0.8 mg/ml of lysozyme (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 10 mg/ml of RNase (Nippon Gene Co., Ltd., Tokyo, Japan), and 80 U/ ml of Lysostaphin (Wako Pure Chemical Industries, Ltd.)) at 37 8C for 12 h, then with 0.5 ml of lysis solution II (0.5 M EDTA (pH 8.0), 1% Sarkosyl, and 0.25 mg/ml of proteinase K (Wako Pure Chemical Industries, Ltd.)) at 50 8C for 24 h. For restriction endonuclease digestion, the plugs were incubated with 30 U of SmaI (Takara Bio Inc., Shiga, Japan) in restriction enzyme buffer at 30 8C for 16 h. DNA fragments were electrophoresed in 1% agarose gel at 6 V/ cm at 14 8C for 22 h with a 3- to 60-s pulse time using CHEF Mapper and CHEF Mapper XA pulsed-field electrophoresis systems (BioRad Laboratories). Lambda Ladder (Bio-Rad Laboratories) was used as a size standard. PFGE patterns were visually compared and evaluated according to the criteria developed by Tenover et al. [28]. Isolates that showed an indistinguishable PFGE pattern were considered as the same strain [18]. 2.6. Statistics Statistical analyses were performed using StatView-J ver. 5.0 (SAS Institute Inc., Cary, NC, USA). The chi-squared test or Fisher’s exact test was used to evaluate the statistical significance of differences. P values of <0.05 were considered statistically significant. 3. Results The overall percentages of children who were under treatment for otitis media, rhinosinusitis, tonsillitis, bronchitis, common cold, and allergic rhinitis were 1.4% (5/363), 4.7% (17/363), 0.6% (2/363), 0.3% (1/363), 0.8% (3/363), and 2.5% (9/363), respectively. No significant difference in morbidity was observed between seasons, DCCs or ages. There were 157 S. pneumoniae isolates from 363 samples during the two seasons (overall carriage rate: 43.3%), and all possessed the lytA gene. Among the 157 isolates, the percentage of each resistance class was as follows: 63.7% (100/157) for penicillinsusceptible S. pneumoniae (PSSP) strains, 35.7% (56/157) for

[()TD$FIG]

K. Hashida et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 664–669

666

70

No. of pbp mutations

0

60

1

2

3

No. of isolates

50 40 30 20 10 0

< = 0.03

0.06

0.12

0.25

0.5

1

2

MIC of penicillin (μg/ml)

Fig. 1. Histogram of penicillin MICs in connection with the number of pbp mutations.

Table 1 summarizes the genetic differences of isolates between seasons, DCCs and age groups. The rates of carriage of S. pneumoniae strains in which any of the pbp mutations and macrolide-resistance genes were detected by PCR were 39.9% (145/363) and 30.9% (112/363), respectively. The rate of nasopharyngeal carriage of any S. pneumoniae strain was not statistically different between the seasons, DCCs, or age groups. However, the rate of carriage of S. pneumoniae with 2 pbp mutations was higher in DCC-A than in DCC-B (32.8% and 17.5%, respectively; P < 0.01), and in children aged 3 years than in those aged 4 years (31.5% and 18.1%, respectively; P < 0.01). The genetic difference of penicillin resistance between isolates from the two age groups was consistent with the result of antimicrobial susceptibility tests; i.e., the percentage of PISP/PRSP strains was significantly higher in children aged 3 years than in those aged 4 years (43.6% (41/94) and 25.4% (16/63), respectively; P < 0.05). On the other hand, the detection rate of the macrolide-resistance genes was not statistically different between the seasons, DCCs, or age groups. The characteristics of the isolated S. pneumoniae strains with different PFGE patterns are listed in Table 2. Fifty different PFGE patterns and, thus, 50 different strains were obtained from among the 157 isolates, and were designated strains S1–S50. Although the number of pbp mutations varied even among isolates of the same strain, every strain except S5 showed a narrow range of penicillin MICs. Meanwhile, the PFGE classification generally corresponded

penicillin-intermediately resistant S. pneumoniae (PISP) strains, 0.6% (1/157) for penicillin-resistant S. pneumoniae (PRSP) strains, 28.7% (45/157) for erythromycin-susceptible S. pneumoniae strains, 1.9% (3/157) for erythromycin-intermediately resistant S. pneumoniae strains, and 69.4% (109/157) for erythromycinresistant S. pneumoniae strains. The percentages of S. pneumoniae strains with the pbp mutation(s) and mefA and/or ermB gene(s) were 92.4% (145/157) and 71.3% (112/157), respectively. Fig. 1 shows a histogram of the correlation between penicillin MICs and the number of pbp mutations. The rate of PSSP strains was significantly higher among the isolates with 1 pbp mutation than among those with 2 pbp mutations (87.7% (57/65) and 46.7% (43/92), respectively; P < 0.05). Fig. 2 shows a histogram of the correlation between erythromycin MICs and macrolide-resistance genes. The rate of erythromycin-susceptible strains was significantly higher among the isolates without the macrolide-resistance genes than among those with the mefA and/or ermB genes (87.0% (39/45) and 5.0% (6/112), respectively; P < 0.01). Conversely, the rate of erythromycin-resistant strains was significantly lower among the isolates without the macrolide-resistance genes than among those with the mefA and/or ermB genes (13.3% (6/45) and 92.0% (103/112), respectively; P < 0.01). There were six isolates with both the mefA and the ermB genes, and their MICs were all 32 mg/ml. These results indicate a close relationship between the MICs and antimicrobial-resistant genes.

[()TD$FIG]

No. of isolates

80

mefA(+) ermB(+)

70

mefA(- ) ermB(+)

60

mefA(+) ermB(-) mefA(-) ermB(-)

50 40 30 20 10 0

< = 0.12

0.25

0.5

1

2

4

MIC of erythromycin (μ g/ml)

8

16

32 < =

Fig. 2. Histogram of erythromycin MICs in connection with macrolide-resistant genes.

K. Hashida et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 664–669

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Table 1 Genetic differences of the isolated S. pneumoniae strains between seasons, DCCs, and age groups. No. of isolates (% of carriage) Season

All S. pneumoniae isolates Isolates with 1 pbp mutation Isolates with 2 pbp mutations Isolates with mefA gene Isolates with ermB gene

DCC

Age

Overall (n = 363)

Summer (n = 181)

Winter (n = 182)

P-value

DCC-A (n = 186)

DCC-B (n = 177)

P-value

3 Year (n = 197)

4 Year (n = 166)

P-value

78 29 49 20 35

79 36 43 14 49

NS NS NS NS NS

85 24 61 15 42

72 41 31 19 42

NS <0.05 <0.01 NS NS

94 32 62 20 45

63 33 30 14 39

NS NS <0.01 NS NS

(43.1) (16.0) (27.1) (11.0) (19.3)

(43.4) (19.8) (23.6) (7.7) (26.9)

(45.7) (12.9) (32.8) (8.1) (22.6)

(40.7) (23.2) (17.5) (10.7) (23.7)

(47.7) (16.2) (31.5) (10.2) (22.8)

(38.0) (19.9) (18.1) (8.4) (23.5)

157 (43.3) 65 (17.9) 92 (25.3) 34 (9.2) 84 (23.1)

NS; not significant.

to the PCR results for the macrolide-resistance gene(s) and erythromycin MICs; isolates of each strain except S11 had basically the same macrolide-resistance gene(s), and those except S5 and S11 mostly belonged to an equal resistance class for erythromycin.

Besides S5 and S11, there were three strains (S1, S2, and S3) with a wide range of erythromycin MICs (0.12–32 mg/ml ), but 17 isolates of S1, 16 isolates of S2, and 9 isolates of S3 showed erythromycin MICs of 8 mg/ml, 32 mg/ml, and 0.12 mg/ml,

Table 2 Characteristics of S. pneumoniae strains with different PFGE patterns. PFGE pattern (n)

S1 (20) S2 (17) S3 (10) S4 (9) S5 (9) S6 (6) S7 (6) S8 (6) S9 (6) S10 (5) S11 (5) S12 (5) S13 (4) S14 (4) S15 (3) S16 (3) S17 (2) S18 (2) S19 (2) S20 (2) S21 (2) S22 (1) S23 (1) S24 (1) S25 (1) S26 (1) S27 (1) S28 (1) S29 (1) S30 (1) S31 (1) S32 (1) S33 (1) S34 (1) S35 (1) S36 (1) S37 (1) S38 (1) S39 (1) S40 (1) S41 (1) S42 (1) S43 (1) S44 (1) S45 (1) S46 (1) S47 (1) S48 (1) S49 (1) S50 (1) n; number of isolates.

pbp mutation(s)

Macrolide-resistance gene(s)

No. of carriers

1

2

Penicillin MIC range (mg/ml)

mefA

ermB

Erythromycin MIC range (mg/ml)

Summer (DCC-A/B)

Winter (DCC-A/B)

6 1 8 7 3 4 6 0 0 3 0 0 4 2 3 0 1 0 2 0 0 1 1 0 1 1 1 0 1 1 1 0 1 1 1 1 1 1 0 0 0 0 0 0 0 1 0 0 0 0

14 16 2 2 6 2 0 6 6 2 5 5 0 2 0 3 1 2 0 2 2 0 0 1 0 0 0 1 0 0 0 1 0 0 0 0 0 0 1 1 1 1 1 1 1 0 1 1 1 1

0.03–0.12 0.06–0.12 0.03–0.12 0.03 0.03–1 0.03 0.03 0.06 1 0.03 0.03–0.06 0.06–0.12 0.03–0.12 0.25 0.03 1 0.03 0.06 0.12–0.5 0.25–1 1 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.06 0.06 0.12 0.12 0.12 0.25 0.25 0.25 0.5 0.5 1 2

0 0 0 0 9 6 0 0 5 0 3 0 3 0 0 0 2 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 1 0 0 0

16 16 1 9 0 6 0 0 0 1 2 0 0 4 3 3 0 2 2 2 2 0 1 0 0 0 1 1 1 0 0 1 1 0 1 0 0 0 0 1 1 0 1 0 1 1 0 1 1 1

0.12–32 0.12–32 0.12–32 8–32  0.25–2 32  0.12 0.12 2–4 0.12 0.12–32 0.12 2–32  2–32  32  4–32  0.5 32  32  32  32  4 32  2 0.12 4 0.25 32  32  0.12 0.12 32  32  0.12 32  0.12 32  0.12 0.12 32  32  32  32  4 32  32  4 32  32  32 

1/1 10/0 0/3 0/3 1/1 0/1 0/0 6/0 0/4 2/0 4/0 5/0 4/0 0/2 0/2 0/2 2/0 0/0 0/1 0/2 0/2 1/0 1/0 1/0 1/0 1/0 1/0 0/0 0/1 0/1 0/1 0/0 0/0 0/0 0/0 0/0 0/0 0/1 1/0 0/1 1/0 1/0 0/0 1/0 0/1 0/0 0/1 0/0 0/1 1/0

18/0 7/0 7/0 0/6 0/7 1/4 0/6 0/0 0/2 3/0 1/0 0/0 0/0 0/2 0/1 0/1 0/0 0/2 0/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1/0 0/0 0/0 0/0 0/1 0/1 0/1 0/1 0/1 0/1 0/0 0/0 0/0 0/0 0/0 1/0 0/0 0/0 0/1 0/0 0/1 0/0 0/0

  



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K. Hashida et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 664–669

respectively. There were 14 strains (S4, S5, S6, S7, S8, S12, S14, S15, S16, S17, S18, S19, S20, and S21) which had identical macrolideresistance gene(s) and showed erythromycin MICs coinciding with the genotype. Thirteen strains (S1, S2, S3, S4, S5, S6, S9, S10, S11, S14, S15, S16, and S19) were observed in both seasons, but only one of these strains (S19) was isolated from the same carrier. Most strains except S3 were observed mostly in only one DCC. Twenty-one strains (42.0%) were isolated from two or more children; 17 of these were each isolated from children belonging to the same DCC. The other four strains (S1, S3, S5, and S6) were found in both DCCs, but showed a seasonal difference in the prevalence of their colonization. Such turnover and transmissibility were independent of the season, children’s age, and genetic mutations. 4. Discussion The overall rate of nasopharyngeal carriage of S. pneumoniae in the present study was 43.3%, slightly lower than that reported by Masuda et al. (53.2%) in Japanese children in a similar setting [29]. The nasopharyngeal carriage rates of S. pneumoniae reported in Europe and North America range from 3.5% to 88.7% among children attending DCCs, living in orphanages, or attending schools in various situations [14,16,19,30–34]. Such a wide dispersion may be explained by the fact that the carriage rate is dependent on multiple factors, such as age, season, the size of the facility, antibiotic treatment, morbidity of acute upper respiratory tract infections, the sampling technique, and the type of infant feeding [35]. The percentage of isolates of PISP/PRSP has been reported to be 0–60.6% among healthy children in Europe and North America [14,19,30,31,33,34] and 21.5–74.1% among patients with community-acquired infections in Japan [25,26,36–39]. Previous studies have also revealed that the percentage of the clinical isolates with 2 pbp mutations ranged from 38.1% to 77.6% in Japan [6,25,36,37,39,40]. In the present study, the percentages of PISP/ PRSP strains and the isolates with 2 pbp mutations were 36.3% (57/157) and 58.6% (92/157), respectively, comparable to the values formerly documented. These data indicate the widespread occurrence of PISP/PRSP in Japan as well as in Western countries. The percentage of isolates of erythromycin-resistant S. pneumoniae was 69.4% (109/157) in our study and has been reported to be 72.0–81.0% among patients with community-acquired infections in Japan [26,38,41,42]. Such percentages are higher than those among healthy children in Western countries, which range between 5.9% and 59.8% [14,19,30,33]. The percentage of clinical isolates with the mefA and/or ermB gene(s) was also high in our results (71.3% (112/157)) as well as in previous surveys in Japan (72.0–87.4%) [26,36,38,40,42]. This phenomenon may be attributable to the prevalent use of macrolides for the treatment of chronic sinusitis and lower respiratory diseases in Japan. The present study demonstrated that there was no significant difference in the rate of S. pneumoniae carriage between the age groups; however, the percentage of PISP/PRSP isolates and the carriage rate of S. pneumoniae isolates with 2 pbp mutations were significantly higher in younger children than in older children. It has been established that children aged 1–2 years are more likely to contract repeated ear infections than older children [43] and that beginning attendance at a DCC at an early age increases the risk of recurrent otitis media [44]. This may suggest that younger children have more of a chance of receiving antibiotic treatment, which would potentially induce resistance genes. Because PFGE analysis does not necessarily reflect all genetic mutations, isolates indistinguishable by PFGE may not be genetically identical. Molecular research with S. pneumoniae strains has revealed that strains with an indistinguishable PFGE

pattern may have heterogeneous mutations in the pbp genes. However, the present study demonstrated a close correlation between the PFGE patterns, macrolide-resistance genes, and erythromycin MICs. Although some discrepancy was observed between the PFGE patterns and pbp mutations, most of the PFGEbased strains showed narrow ranges of penicillin MICs. These observations suggest that molecular typing by PFGE is a useful tool for the epidemiological investigation of S. pneumoniae. Several previous studies have used PFGE to investigate the molecular epidemiology, dynamics, and variable genetic heterogeneity of S. pneumoniae colonizing the upper respiratory tracts of children [14,16,18,19]. Sa´-Lea˜o et al. [19] observed two types of turnover/transmission of S. pneumoniae in a DCC setting in their longitudinal study; one showed a high capacity for prolonged colonization in the host, but the other had a low capacity for prolonged colonization with a high degree of transmissibility. Sulikowska et al. [14] conducted bacterial sampling from the nasopharynges of children in a DCC or an orphanage in winter and in the following spring, and found that only 3 out of 21 children were colonized by the same or closely related strains by PFGE analysis. In the present study, 13 strains were observed in both seasons, but only one of these strains was isolated from the same carrier. We also found that more than 40% of the strains colonized two or more children, who mostly belonged to the same DCC. These findings suggest the frequent occurrence of vigorous genetic turnover and horizontal transmission of S. pneumoniae among healthy children in DCCs. In conclusion, we investigated the characteristics of nasopharyngeal S. pneumoniae carriage in children attending DCCs by means of antibiotic susceptibility testing, PCR, and PFGE in the summer of 2004 and in the following winter. S. pneumoniae was widely spread among healthy children, and the percentages of strains with antibiotic-resistance gene(s) and antibiotic-resistant strains were considerably high. We also observed vigorous genetic turnover and horizontal transmission of S. pneumoniae among these children. Hygienic and prophylactic countermeasures against this health problem need to be taken without delay.

Acknowledgement This research was partially supported by the Ministry of Education, Science, Sports and Culture through a Grant-in-Aid for Young Scientists (B) (16791032, 2004).

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