Prevalence of Streptococcus Pneumoniae Isolates Bearing Macrolide Resistance Genes in Association with Integrase Genes of Conjugative Transposons in Japan T

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ORIGINAL ARTICLE 10.1111/j.1469-0691.2005.01232.x

Prevalence of Streptococcus pneumoniae isolates bearing macrolide resistance genes in association with integrase genes of conjugative transposons in Japan T. Shiojima1, Y. Fujiki2, H. Sagai2, S. Iyobe3 and A. Morikawa1

1Department of Pediatrics, Gunma University School of Medicine, Maebashi, 2Pharmaceuticals Scientiﬁc & Technical Department, Asahi Kasei Pharma Corporation, Tokyo and 3Graduate School of Medical Sciences, Kitasato University, Sagamihara, Japan

ABSTRACT The relationship between susceptibility to macrolides and tetracycline, and the distribution of the resistance genes erm(B), mef(A) and tet(M) and the integrase gene of the conjugative transposon, Int-Tn, was examined in 43 isolates of Streptococcus pneumoniae from Gunma Prefecture, Japan. Thirty-ﬁve isolates were resistant to both macrolides and tetracycline and carried tet(M); erm(B) and mef(A) were detected in 19 and 16, respectively, of these isolates. Sixteen mef(A)-positive isolates were all identiﬁed as mef(E) variants. Int-Tn of Tn1545 was associated with 17 erm(B)- and 14 mef(A)-bearing isolates, and Int–Tn of Tn5252 was found in the remaining two mef(A)-carrying isolates. Pneumococcal strains with resistance to macrolides conferred by erm(B) or mef(A) in association with the integrase gene of conjugative transposon Tn1545 or Tn5252 appear to be prevalent in Japan. Keywords Conjugative transposons, erm(B), macrolides, mef(A), resistance, Streptococcus pneumoniae Original Submission: 5 October 2004; Revised Submission: 18 March 2005; Accepted: 2 May 2005 Clin Microbiol Infect 2005; 11: 808–813

occurs with 14-membered (e.g., erythromycin and INTRODUCTION clarithromycin) or 15-membered (e.g., azithromy- Streptococcus pneumoniae is one of the most cin) macrolides, lincosamides and streptogramin important bacterial pathogens causing respiratory B. In contrast, rokitamycin, a 16-membered tract infections such as community-acquired macrolide, does not induce gene expression in pneumonia and otitis media, bacteraemia and many resistant strains carrying erm(B) [4,5]. The meningitis. Treatment of pneumococcal infections second mechanism involves an active efflux is becoming difficult because of dissemination of pump, which removes 14- and 15-membered penicillin-resistant strains and the rapid develop- macrolides, but not 16-membered macrolides, ment of resistance to other antibiotics, including from bacterial cells. This efflux pump is encoded macrolides [1]. by mef(A), and isolates carrying mef(A) are sus- There are two principal mechanisms of macro- ceptible to rokitamycin, lincosamides and strep- lide resistance, namely target site modification togramin B [4,5]. and active efflux. Target site modification is The recent emergence of multiple-antibiotic mediated by a methylase, which methylates a resistance in S. pneumoniae has been associated specific adenine residue on 23S rRNA. In pneu- with the conjugative transposon Tn1545, which mococci, this enzyme is encoded by erm(B) [2,3], confers resistance to tetracycline, kanamycin which is expressed in either an inducible or a and macrolides, encoded by tet(M), aphA-3 and constitutive manner. Active induction of erm(B) erm(B), respectively, and is self-transmissible to a wide variety of Gram-positive bacteria [6]. Another conjugative transposon, Tn5252, which Corresponding author and reprint requests: T. Shiojima, confers chloramphenicol resistance, has also been Department of Pediatrics, Gunma University School of Medi- cine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan found in S. pneumoniae [7]. Conjugative trans- E-mail: [email protected] posons contain an Int-Tn gene, which encodes an

2005 Copyright by the European Society of Clinical Microbiology and Infectious Diseases Shiojima et al. S. pneumoniae macrolide resistance genes 809 integrase that is essential for the integration of the primers were as follows: for erm(B),5¢-CGTACCTTGGAT- ¢ ¢ genetic element into chromosomal DNA during ATTCACCG-3 (forward) and 5 -GTAAACAGTTGACGA- TATTCTCG-3¢ (reverse) [13]; for mef(A),5¢- GGGACCTGC- conjugation. Since the Int-Tn sequence of Tn1545 CATTGGTGTGC-3¢ (forward) and 5¢-CCCAGCTTAGGTA- (Int-Tn1545) differs from the Int-Tn sequence of TACGTAC-3¢ (reverse) (this article); and for tet(M),5¢-GA- Tn5252 (Int-Tn5252), it is possible to design PCR ACTGTATCCTAATGTGTG-3¢ (forward) and 5¢-GATACTC- primer sets targeted at each sequence [8,9]. TAACCGAATCTCG-3¢ (reverse) [14]. To distinguish mef(A) ¢ ¢ The present study investigated the susceptibil- from mef(E), primers 5 -TGGTTCGGTGCTTACTATTGT-3 (forward) and 5¢-CCCCTATCAACATTCCAGA-3¢ (reverse) ity of S. pneumoniae isolates in Japan to macro- were used for mef(A), and 5¢-GGGAGATGAAAGAA- lides, tetracycline and other antibiotics. The GGAGT-3¢ (forward) and 5¢-TAAAATGGCACCGAAAG-3¢ distribution of resistance genes and integrase (reverse) were used for mef(E) [10]. To detect Int-Tn genes, ¢ ¢ genes was examined by PCR, using primer sets primers 5 -GCTCAAAGACCAAAGGTTAGAA-3 (forward) and 5¢-TGTGTGGCAATTTATCCTCGTT-3¢ (reverse) were specific for tet(M), erm(B), mef(A), Int-Tn1545 and used for Int-Tn1545 [8], and primers 5¢-TTAAGTTCTCTCC- Int-Tn5252, and serotypes were determined. In TATGACTGC-3¢ (forward) and 5¢-AAGTCTTTGAACTCA- addition, the presence of mef(A) and mef(E) var- TCGACAC-3¢ (reverse) were used for Int-Tn5252 [9]. Each iants [5] was determined using specific PCR 50-lL reaction mixture contained 5 lL of template DNA, 10 lL l primers [10]. (10 pmol) of each forward and reverse primer, and 25 Lof PCR reaction kit mixture (Takara, Tokyo, Japan). Gene amplification was performed using a PC-700 thermal cycler (Astec, MATERIALS AND METHODS Tokyo, Japan) at 94 C for 5 min, followed by 35 cycles of 94 C for 1 min, 50Cor54C for 1 min, and 72C for 1 min Bacterial isolates [8–10,13,14]. Between January and April 2002, 43 non-duplicate clinical isolates of S. pneumoniae were collected from nine medical Serotyping centres in Gunma Prefecture, Japan, from sputum, pharyngeal Serotyping was performed by agglutination with polyvalent swab, nasal mucus and other specimens. Identification was and monovalent antisera for S. pneumoniae capsules (Denka based on Gram’s stain, colonial morphology and haemolysis Seiken, Tokyo, Japan). One drop of each antiserum was placed on blood agar medium, susceptibility to optochin disks and on a glass slide and mixed with a visible amount of detection of an autolysin gene by PCR [11]. pneumococcal cells. Physiological saline was used as a control instead of an antiserum. Antibiotics and susceptibility tests The antibiotics used were erythromycin, clarithromycin, RESULTS azithromycin, rokitamycin, lincomycin, chloramphenicol and tetracycline. MICs were determined by agar dilution [12] Detection of macrolide resistance genes by PCR using Muller–Hinton agar (Difco, Tokyo, Japan) supplemen- and susceptibility testing ted with horse serum 5% v ⁄ v. Inocula were prepared from cells grown freshly in brain–heart infusion broth, with PCR revealed that 19 (44.2%) and 16 (37.2%)of c. 104 CFU ⁄ spot. the 43 isolates of S. pneumoniae carried erm(B) and mef(A), respectively. Eight (18.6%) isolates did not Detection of macrolide and tetracycline resistance genes and integrase genes of conjugative transposons by PCR carry any resistance gene, and no isolate carried both genes (Table 1). Typical PCR amplification Genomic DNA was extracted from each isolate by incubating products of mef(A) and erm(B) are shown in Fig. 1 bacterial cell suspensions at 60C for 10 min, followed by 90C for 5 min; each preparation was then assayed for the presence (lanes 1 and 2). Further PCRs with primer sets of erm(B), mef(A) and tet(M) by PCR. The oligonucleotide specific for either mef(A) or mef(E) [10] revealed

Table 1. Antibiotic resistance patterns, genotypes and serotypes (or serogroups) for 43 Streptococcus pneumoniae isolates from Japan

Genotype Serotype (or serogroup)

Resistance pattern erm(B) mef(A) tet(M) Int-Tn1545 Int-Tn5252 3691419222335NTTotalno.

Mac, Lcm, TET, CHL + – + + – 7 1 2 10 Mac, Lcm, TET + – + + – 1 1 1 4 7 Mac, Lcm, TET + – + – – 2 2 Mac, TET – + + + – 4 1 8 1 14 Mac, TET – + + – + 2 2 Susceptible –––– – 2 2 1 2 1 8

Mac, macrolides; Lcm, lincomycin; TET, tetracycline; CHL, chloramphenicol; NT, non-typeable.

2005 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 11, 808–813 810 Clinical Microbiology and Infection, Volume 11 Number 10, October 2005

M 1234 All 16 isolates carrying mef(A) were intermediately-resistant to erythromycin, clarithromycin and azithromycin, susceptible to rokitamycin, lincomycin and chloramphenicol, and resistant to tetracycline (Fig. 2). The MICs of kanamycin for the 43 isolates ranged from 32 to 128 mg ⁄ L, indicating that they did not acquire a gene conferring kanamycin resistance (data not shown).

Detection of tet(M) and Int-Tn by PCR 800 bp The antibiotic resistance patterns and genes for antibiotic resistance and conjugation are shown in 500 bp Table 1. The tet(M) gene was detected in 35 isolates, which were resistant to tetracycline and carried either erm(B) or mef(A), but was not 300 bp amplified from isolates susceptible to tetracycline and macrolide. Int-Tn1545 was amplified from 31 (72.1%)of the 43 isolates. Among these 31 isolates, ten were resistant to macrolides, lincomycin, tetracycline and chloramphenicol, and seven were resistant to macrolides, lincomycin and tetracycline; these 17 isolates carried erm(B). The remaining 14 isolates were resistant to macrolides and tetracycline, and Fig. 1. Examples of amplicons obtained following specific PCRs for detection of drug resistance and Int-Tn genes. carried mef(A) (Table 1). PCR was performed for Lanes: M, size marker; 1, mef(A), 402 bp; 2, erm(B), 224 bp; Int-Tn5252, using DNA prepared from four iso- 3, Int-Tn1545, 742 bp, and tet(M), 377 bp; 4, Int-Tn5252, lates carrying tet(M) and either erm(B) or mef(A), 877 bp. but not Int-Tn1545. Int-Tn5252 was detected in two isolates carrying mef(A) (Table 1). Represen- tative amplicons for tet(M), Int-Tn1545 and Int- that all 16 mef(A)-carrying isolates were mef(A)- Tn5252 are shown in Fig. 1. negative but mef(E)-positive; i.e., they were mef(E) variants. Serotyping The relationship between antibiotic susceptibility and macrolide resistance genes is shown in Serotypes for the isolates are shown in Table 1. Fig. 2. Eight isolates in which no macrolide The isolates belonged to serotype 3 (n = 8), resistance gene was detected by PCR were serogroups 6 (n = 10) and 9 (n = 1), serotype 14 susceptible to all of the antibiotics examined. (n = 1), and serogroups 19 (n = 10), 22 (n = 1), 23 Among the 19 isolates carrying erm(B), five were (n = 5) and 35 (n = 2), with five isolates being intermediately-resistant to erythromycin, while non-typeable. Among the eight isolates belonging the remaining 14 were highly-resistant (MICs to serotype 3, seven were resistant to chloram- > 128 mg ⁄ L). Eight and 13 isolates showed high- phenicol and all carried erm(B). Serogroup 19 was level resistance to clarithromycin and azithromy- not found among the 19 erm(B)-carrying isolates, cin, respectively. One isolate was highly-resistant whereas eight of 16 mef(A)-carrying isolates to rokitamycin, 12 were intermediately-resistant belonged to serogroup 19 (Table 1). and six were susceptible. Levels of resistance to lincomycin were slightly higher than those for DISCUSSION erythromycin or azithromycin. All of the erm(B) isolates were resistant to tetracycline, and ten Macrolide resistance in S. pneumoniae has were resistant to chloramphenicol (Fig. 2). increased in European countries, e.g., Italy,

2005 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 11, 808–813 Shiojima et al. S. pneumoniae macrolide resistance genes 811

25 Erythromycin 25 Clarithromycin

20 20

15 15

10 10

5 5

0 0 ≤ 0.1250.25 0.5 1 2 4 8 16 32 64 128 >128 ≤ 0.125 0.25 0.5 1 2 4 8 16 32 64 128 >128 25 25 Azithromycin Rokitamycin

20 20

15 15

10 10

5 5

0 0 ≤ 0.125 0.25 0.5 1 2 4 8 16 32 64 128 >128 ≤ 0.125 0.25 0.5 1 2 4 8 16 32 64 128 >128 25 25 Lincomycin Tetracycline Number of strains 20 20

15 15

10 10

5 5

0 0 ≤ 0.125 0.25 0.5 1 2 4 8 16 32 64 128 >128 ≤ 0.125 0.25 0.5 1 2 4 8 16 32 64 128 >128 25 MIC (mg/L) Chloramphenicol

15 no resistance : 8 strains

erm (B) : 19 strains 10 mef (A) : 16 strains 5 Fig. 2. Distribution of the erm(B) and mef(A) macrolide resistance 0 genes according to MICs of various ≤ 0.125 0.25 0.5 1 2 4 8 16 32 64 128 >128 antibiotics. MIC (mg/L)

France and Spain, as well as in North America. being resistant to 14-membered macrolides In Asia, the incidence of erythromycin-resistant (erythromycin and clarithromycin) and a 15- S. pneumoniae has risen in Hong Kong, Taiwan membered macrolide, azithromycin. In contrast, and Japan to nearly 80% [15]. A high incidence 70% (30 ⁄ 43) of isolates were susceptible to of macrolide resistance was conﬁrmed in the rokitamycin, a 16-membered macrolide used present study, with 81.4% (35 ⁄ 43) of the isolates clinically in Japan, which is expected to be used

2005 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 11, 808–813 812 Clinical Microbiology and Infection, Volume 11 Number 10, October 2005 widely for the treatment of pneumococcal 16 isolates in the present study were identified as infections. mef(E), and most of these isolates belonged to Wide geographical differences in the distribu- serogroups 19 (n = 8) and 6 (n = 6). These find- tion of erm(B) and mef(A) throughout the world ings suggest worldwide dissemination of macro- have been reported [16]. Nishijima et al. [17] lide-resistant S. pneumoniae carrying mef(E) which reported that isolates carrying erm(B), mef(A) or belong to serogroup 19. both genes comprised 44%,40% and 16%, respectively, of erythromycin-resistant isolates REFERENCES from Japan in 1995. In the present study, 54% of erythromycin-resistant isolates carried erm(B), 1. Appelbaum PC. 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