Jpn. J. Infect. Dis., 61, 454-456, 2008 Original Article T Serotypes and Antimicrobial Susceptibilities of Group A Streptococcus Isolates from Pediatric Pharyngotonsillitis Keiji Funahashi*, Kazumasa Nakane, Naoko Yasuda, Michio Suzuki1, Atushi Narita1, Naoko Arai1, Jaekun Ahn1, Norio Koyama1, Hajime Ushida1, Naoko Nishimura1 and Takao Ozaki1 Department of Clinical Laboratory and 1Department of Pediatrics, Konan Kosei Hospital, Aichi 483-8704, Japan (Received April 1, 2008. Accepted August 25, 2008) SUMMARY: Group A streptococcus (GAS) is a major cause of pediatric pharyngotonsillitis. In this study we determined the T serotype and antimicrobial susceptibility of GAS isolates from Japanese children. From January to December 2006, a total of 438 isolates of GAS were obtained from pharyngeal swabs of 438 children with pharyngotonsillitis. The commonest T serotype was type 1 (110 strains, 25.1%), followed by type 12 (107, 24.4%) and type 4 (77, 17.6%). All GAS isolated from pharyngeal swabs were susceptible to β-lactams (benzylpenicillin, amoxicillin, cefotaxime, ceftriaxone, imipenem, panipenem, and cefditoren) and vancomycin, but 19.6, 19.6, 3.2, 11.6, and 27.6% were resistant to erythromycin, clarithromycin, clindamycin, minocycline, and norfloxacin, respectively. Resistance varied considerably with the T serotype. In particular, type 4 isolates had the highest resistance (67.5, 67.5, 26.0, and 53.2% were resistant to erythromycin, clarithromycin, minocycline, and norfloxacin, respectively). bated on Trypticase Soy Agar II plates with 5% sheep blood INTRODUCTION (Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) for 24 Group A streptococcus (GAS) is a major causative agent h. Colonies with diameters between 1 and 2 mm showing of pharyngotonsillitis and impetigo, which can be transmitted β hemolysis on culture media were identified, and pure from human to human. Droplet transmission in a communal cultures were then performed on the same culture media. living setting such as schools and nursery centers or within a Lancefield serogrouping (4) was performed using Streptex family is the major route of GAS transmission (1). Further- (Remel Inc., Lenexa, Kans., USA) to identify GAS. more, GAS is known to occasionally cause group infections. T serotyping: T serotyping was performed using GAS T- Since the late 1970s, the incidences of acute glomerulone- typing antisera (Denka Seiken Co., Ltd., Tokyo, Japan). phritis and rheumatic fever, which are important complica- Antimicrobial susceptibility testing: The minimal inhibi- tions of GAS pharyngotonsillitis, have decreased markedly. tory concentrations (MICs) of 14 antimicrobial agents, i.e., However, in the late 1980s, cases of streptococcal toxic shock benzylpenicillin (PCG), amoxicillin (AMPC), cefotaxime syndrome (STSS), a septic condition associated with fatal (CTX), ceftriaxone (CTRX), cefditoren (CDTR), cefcapene multiple organ failure, were reported in several places, draw- (CFPN), panipenem (PAPM), imipenem (IPM), erythromy- ing renewed attention to GAS infections (2,3). cin (EM), clarithromycin (CAM), clindamycin (CLDM), To understand the current status of GAS, we investigated minocycline (MINO), norfloxacin (NFLX), and vancomycin T serotypes and antimicrobial susceptibilities of GAS iso- (VCM), were measured by the broth microdilution method. lates obtained from pediatric patients with pharyngotonsilli- Isolates were determined to be susceptible (S), intermediate tis. (I), or resistant (R) with respect to each agent according to the criteria of the Clinical and Laboratory Standards Insti- tute (CLSI) (5). Because there were no CLSI streptococcal MATERIALS AND METHODS breakpoints for MINO or NFLX, we substituted those of Sample collection: Our study targets were 438 GAS strains tetracycline and levofloxacin, respectively. Isolates classified isolated from pharyngeal swabs obtained from 438 patients into category I or R were analyzed as resistant strains. with pharyngotonsillitis (mean age, 6.0 years; range, 6 months to 15.8 years) who visited the pediatric department of Konan RESULTS Kosei Hospital from January to December 2006. The strains re-isolated within 3 months after the first isolation from a T serotypes: Among 438 strains examined, the T serotype single patient were excluded from the analysis, since they most frequently isolated was type 1 (110 strains, 25.1%), fol- would likely be identical to strains already collected. lowed by type 12 (107 strains, 24.4%) and type 4 (77 strains, Isolation and identification of GAS: Pharyngeal swabs 17.6%). These three epidemic forms accounted for 67.1% of obtained from patients with pharyngotonsillitis were incu- the total isolates. Antimicrobial susceptibility: The MICs of PCG, AMPC, *Corresponding author: Mailing address: Department of Clinical CTX, CTRX, CDTR, CFPN, PAPM, and IPM for all strains Laboratory, Konan Kosei Hospital, 137 Ohmatsubara, Takaya- were ≤0.12 μg/mL. All strains were susceptible to β-lactams. cho, Konan-shi, Aichi 483-8704, Japan. Tel: +81-587-51-3333, In addition, all strains showed high susceptibilities to CDTR, Fax: +81-587-51-3300, E-mail: [email protected]. CFPN, PAPM, and IPM. On the other hand, 86 (19.6%), 86 or.jp (19.6%), 14 (3.2%), 51 (11.6%), and 121 (27.6%) strains 454 Table 1. Cross resistance rates for 5 antimicrobials Cross resistance rates Antimicrobial Resistant strains Resistant strains Resistant strains Resistant strains Resistant strains to EM (n = 86) to CAM (n = 86) to CLDM (n = 14) to MINO (n = 51) to NFLX (n = 121) EM – 100% (86/86) 100% (14/14) 27.5% (14/51) 27.3% (33/121) CAM 100% (86/86) – 100% (14/14) 27.5% (14/51) 27.3% (33/121) CLDM 16.3% (14/86) 16.3% (14/86) – 21.6% (11/51) 0% (0/121) MINO 16.3% (14/86) 16.3% (14/86) 78.6% (11/14) – 19.0% (23/121) NFLX 38.4% (33/86) 38.4% (33/86) 0% (0/14) 49.0% (25/51) – EM, erythromycin; CAM, clarithromycin; CLDM, clindamycin; MINO, minocycline, NFLX, norfloxacin. Table 2. Major T serotypes and antimicrobial resistance rates T serotype Antimicrobial 1 (n = 110) 4 (n = 77) 12 (n = 107) EM 3.6% (4/110) 67.5% (52/77) 19.6% (21/107) CAM 3.6% (4/110) 67.5% (52/77) 19.6% (21/107) MINO 3.6% (4/110) 26.0% (20/77) 10.3% (11/107) CLDM 0.9% (1/110) 0% (0/77) 11.2% (12/107) NFLX 22.7% (25/110) 53.2% (41/77) 6.5% (7/107) Abbreviations are in Table 1. Table 3. Resistance rates of GAS isolates obtained from pediatric patients with pharyngotonsillitis at Konan Kosei Hospital between 1996 and 2006 Years that the surveys were conducted Antimicrobial 1996* (n = 431) 2001* (n = 317) 2003* (n = 295) 2006 (n = 438) EM 8.6% (37/431) 13.6% (43/317) 20.0% (59/295) 19.6% (86/438) CAM 13.2% (42/317) 19.7% (58/295) 19.6% (86/438) CLDM 1.6% (5/317) 5.4% (16/295) 3.2% (14/438) MINO 13.2% (57/431) 6.0% (19/317) 10.5% (31/295) 11.6% (51/438) *: Data in 1996 from reference (6), 2001 from reference (7), and 2003 from reference (8). Abbreviations are in Table 1. were resistant to EM, CAM, CLDM, MINO, and NFLX, face of GAS. In a serotype survey, the typing of M-protein respectively. Cross resistance was seen among the five anti- (which is a virulence factor) is important, but in Japan, anti- microbials, except for between CLDM and NFLX (Table 1). sera for M typing are not commercially available. On the other The strains resistant to EM were the same as the strains resist- hand, T typing is widely used as a survey tool, although T- ant to CAM, and the 14 strains resistant to CLDM were all protein is not a virulence factor, because it can be performed resistant to both EM and CAM. with commercially available antisera, correlates with M typ- T serotypes and resistance rates to antimicrobial agents: ing to some extent, is not affected by subculture, and is rela- Table 2 shows the three major T serotypes and their resist- tively easy to perform (11,12). T1, T4, and T12 account for ance rates to antimicrobial agents. Antimicrobial suscepti- the majority of the T serotypes of GAS isolates in Japan (11). bilities varied depending on the T serotype. In particular, type Our present results showed the isolation of T1, T12, and T4 4 serotype tends to be highly resistant, with 67.5, 67.5, 26.0, in decreasing order of frequency, and approximately agreed and 53.2% showing resistance to EM, CAM, MINO, and with the results of the nationwide survey. NFLX, respectively. Penicillins and macrolides are recommended for the treat- Changes in resistance rates to antimicrobial agents: We ment of GAS pharyngotonsillitis (1,13). However, macrolide- previously conducted three surveys of the same type as the resistant GAS strains have increased worldwide (14), and one present survey (6-8), allowing us to make comparisons of possible cause of such resistance is a mutation at position resistance rates over time. The number of strains showing 2058 of 23S rRNA. In the present study, there were no strains resistance to EM and CAM have gradually increased since with resistance to penicillin, cephem, and carbapenem anti- 1996, and these two currently account for approximately 20% bacterials, or to VCM, but the rates of resistance to EM and of total strains. Resistance rates to CLDM and MINO changed CAM or NFLX were as high as 20 and 28%, respectively. within ranges of 1.6-5.4% and 6.0-13.2%, respectively (Table The resistance of serotype T4 to EM, CAM, MINO, and 3). NFLX was high. We have previously performed similar surveys three times (in 1996, 2001, and 2003) (6-8). The iso- lation rate of type 12 was high in all of the surveys, including DISCUSSION the present one, but the isolation rates of other types have Pediatric pharyngotonsillitis is one of the diseases that we varied between surveys.