Antibiotic susceptibility according to genotype of penicillin-binding protein and macrolide resistance genes, and serotype of Streptococcus pneumoniae isolates from community-acquired pneumonia in children

Naoko Chiba1, Reiko Kobayashi1, Keiko Hasegawa1, Miyuki Morozumi1, Eiichi Nakayama1,2, Takeshi Tajima2, Satoshi Iwata3, Kimiko Ubukata1,* on behalf of the Acute Respiratory Diseases Study Group

1 Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan; 2 Department of Pediatrics, Hakujikai Memorial Hospital, Adachi-ku, Tokyo, 123-0864, Japan; 3 Department of Pediatrics, Tokyo National Medical Center, Higashigaoka, Meguro-ku, Tokyo, 152-8902, Japan


* Corresponding author. Tel: +81-3-5791-6385; Fax: +81-3-5791-6386; E-mail: ubukatak{at}lisci.kitasato-u.ac.jp

Received 29 April 2005; returned 19 May 2005; revised 14 June 2005; accepted 25 July 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Antibiotic susceptibilities, genes mediating ß-lactam and macrolide resistance, and serotypes were analysed for strains of Streptococcus pneumoniae.

Methods: A total of 392 strains of S. pneumoniae were isolated from paediatric patients with community-acquired pneumonia between May 2002 and 2004. All strains were classified into six genotype patterns according to the mutations found in the pbp1a, pbp2x and pbp2b genes identified by PCR. These results are represented by adding ‘g’, indicating genotypic identification.

Results: Thirty-nine per cent of the isolates showed mutations in either one or two PBP genes (gPISP, where PISP stands for penicillin-intermediate resistant S. pneumoniae) and 52.3% had mutations in three genes (gPRSP, where PRSP stands for penicillin-resistant S. pneumoniae). The majority of the strains had a macrolide resistance gene: mef(A), (30.6%); erm(B), (48.5%); or both mef(A) and erm(B), (7.7%). The most frequent serotypes of these strains were: 6B (23.2%), 23F (17.6%), 19F (17.3%), 14 (10.5%) and 6A (8.2%). Serotypes of the seven-valent conjugate vaccine covered 70.9% of all isolates, and 89.8% of gPRSP. Serotypes of the strains with cefotaxime MICs of ≥2 mg/L were almost all of a vaccine type.

Conclusions: The results suggest that introduction of conjugate vaccines into infants and children is necessary for the prevention of pneumococcal infections in Japan.

Keywords: S. pneumoniae , antibiotic resistance genes , PBPs , respiratory tract infections


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae is a major pathogen associated with respiratory tract infections (RTIs), acute otitis media, septicaemia, and meningitis acquired in the community.

In Japan, penicillin non-susceptible S. pneumoniae has been increasing rapidly since around 1990. According to a nationwide surveillance study that was conducted between 1998 and 2000, the prevalence of penicillin-intermediate resistant S. pneumoniae (PISP) and penicillin-resistant S. pneumoniae (PRSP) was 34.4 and 49.0%, respectively.1 In parallel, macrolide resistance increased to 70%.2

ß-Lactam resistance in S. pneumoniae is mediated mainly by multiple genes encoding the PBP1A, PBP2X and PBP2B enzymes (where PBP stands for penicillin-binding protein), which are involved in peptidoglycan synthesis.3 We have developed a rapid identification method using PCR to detect mutations in these genes.4

Conjugate vaccines have recently attracted attention for prevention of various pneumococcal infections5,6 and large-scale clinical trials of the 7- or 11-valent vaccine were conducted in several countries. As a result, some countries have already approved the use of this vaccine in children.

In the present paper, we describe antibiotic susceptibility and identification of resistance genes for S. pneumoniae from paediatric patients with pneumonia, and estimate the coverage rates of 7- and 13-valent conjugate vaccines in Japan.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strains

The acute respiratory diseases (ARD) study was made possible by participation of paediatric physicians belonging to 10 medical institutions between May 2002 and 2004. A total of 392 strains of S. pneumoniae were isolated from clinical samples of infants and children (n = 1121) with community-acquired pneumonia (CAP). Identification of the isolates as S. pneumoniae was confirmed by PCR for the autolysin (lytA) gene in our laboratory (Kitasato Institute for Life Sciences, Kitasato University).

Susceptibility testing

MICs of ß-lactam and macrolide antibiotics were determined by an agar dilution method using Mueller–Hinton agar (MH; Difco Laboratories) supplemented with 5% defibrinized sheep blood. Antibiotics employed in this study were: penicillin and ampicillin (Meiji Seika Kaisha Ltd, Tokyo, Japan); cefotaxime and telithromycin (Aventis Pharma Ltd, Tokyo, Japan); and meropenem (Sumitomo Pharmaceuticals Co., Ltd, Osaka, Japan). S. pneumoniae ATCC 49619 was used as a quality control strain for susceptibility testing.

Serotyping

Serotypes of S. pneumoniae strains were determined by the Quellung reaction using antiserum purchased from the Statens Serum Institut (Copenhagen, Denmark).

Identification of antibiotic resistance genes by PCR

Oligonucleotide primers for detection of three PBP genes and macrolide resistance genes, mef(A) and erm(B), were used as previously described.2

Each reaction mixture contained (i) primers for detecting the lytA and pbp1a genes, (ii) primers for detecting the pbp2x and pbp2b genes, or (iii) primers for detecting the mef(A) and erm(B) genes. PCR conditions were also described previously.4

Sequencing

A total of 28 strains showing MICs of ≥4 mg/L for ampicillin or ≥2 mg/L for cefotaxime were termed tentatively high-resistant PRSP (H-PRSP), and the DNA region corresponding to the transpeptidase domain in each PBP gene was amplified. The nucleotide sequences were determined with an ABI PRISM 377 DNA sequencer.

PFGE analysis

PFGE analysis was carried out by a modification of the method described previously.7

For restriction endonuclease digestion, the plugs were incubated in restriction enzyme buffer with 20 U of ApaI at 37°C for 16 h. Electrophoresis was performed with a CHEF Mapper (Bio-Rad Laboratories, Hercules, CA, USA). Separation of fragments was carried out at 5.7 V/cm at 14°C for 18 h.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All 392 strains were firstly classified into the following six genotypes by PCR for pbp1a, pbp2x, and pbp2b genes: (i) gPSSP with three normal genes (n = 33, 8.4%); (ii) gPISP (pbp2x) with abnormal pbp2x (n = 72, 18.4%); (iii) gPISP (pbp2b) with abnormal pbp2b (n = 2, 0.5%); (iv) gPISP (pbp2x + 2b) with abnormal pbp2x and pbp2b (n = 26, 6.6%); (v) gPISP (pbp1a + 2x) with abnormal pbp1a and pbp2x (n = 54, 13.8%); (vi) gPRSP with three abnormal PBP genes (n = 205, 52.3%).

The MIC ranges and MIC50s of four ß-lactam antibiotics against gPRSP were as follows: penicillin, 0.5–4 and 2 mg/L; ampicillin, 0.25–8 and 2 mg/L; cefotaxime, 0.25–8 and 0.5 mg/L; meropenem, 0.063–1 and 0.5 mg/L.

Although strains of gPISP (pbp2x) exhibited negligible resistance to ampicillin with a 2-fold increase over the MIC50 of gPSSP (0.031 mg/L), the MIC50 of cefotaxime was 16-fold higher than that of gPSSP (0.016 mg/L). MIC50s of ampicillin and cefotaxime also varied in gPISP (pbp2x + 2b) (0.5 and 0.25 mg/L) and gPISP (pbp1a + 2x) (0.25 and 1 mg/L). Cefotaxime MICs against gPISP (pbp1a + 2x) increased markedly, and were also affected by abnormal pbp1a genes. Susceptibilities to ampicillin were equally influenced by abnormal pbp1a, pbp2x and pbp2b genes.

Along with the above results, it was noteworthy that some strains showed an ampicillin MIC of ≥4 mg/L (n = 13) or a cefotaxime MIC of ≥2 mg/L (n = 15). Five PBP genes in these H-PRSP strains were analysed in detail, as described below.

For macrolide resistance, the numbers of strains possessing mef(A), erm(B) or both genes were 120 (30.6%), 190 (48.5%) and 30 (7.7%), respectively. The number of susceptible strains without any resistance gene was as low as 52 strains (13.3%). The MICs of telithromycin for the strains with erm(B) or mef(A) were from 0.063 to 0.125 mg/L, except for four (1.0%) strains with MICs of 2–4 mg/L.

Capsular serotypes of tested strains are listed in Table 1 according to the genotypic patterns for PBP genes.


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Table 1. Serotype distribution according to genotype patterns classified by PCR for pbp genes in S. pneumoniae isolates

 
The most frequent serotypes were: 6B (23.2%), 23F (17.6%), 19F (17.3%) and 14 (10.5%). The disproportionate distributions of serotypes of gPSSP, gPISP and gPRSP were confirmed ({chi}2 = 595.9100, P = 0.0000). Various serotypes were also noted in gPISP (pbp2x) strains, of which serotypes 6A, 6B and 3 were most prevalent. Serotypes 23A and 14 showed the greatest prevalence in gPISP (pbp2x + 2b) and gPISP (pbp1a + 2x) strains, respectively. Five serotypes (19F, 6B, 23F, 6A and 14) accounted for 96.1% of gPRSP, while there were only three strains each of serotypes 23A and 35.

Incidentally, 7- and 13-valent conjugate vaccines covered 70.9 and 84.9% of all strain serotypes tested, respectively. However, being confined to gPRSP, the coverage rates of 7- and 13-valent conjugate vaccines were as high as 89.8 and 96.1%, respectively.

DNA sequences of transpeptidase regions in pbp1a, pbp1b, pbp2a, pbp2x and pbp2b genes were determined for H-PRSP strains. The results were compared with the sequences of gPRSP and the R6 strain.

Table 2 shows the results obtained from 16 strains that had amino acid substitutions different from those found in the major gPRSP. Among strains with an ampicillin MIC of ≥4 mg/L, 10 amino acid substitutions near the conserved motif Lys614-Thr615-Gly616 (KTG) in the pbp2b gene, as described by Kosowska et al.8 were observed in only one strain (ARD-963) with an MIC of 8 mg/L which were different from those found in other strains; substitutions of Ala591->Ser, Gly596->Pro, Asn605->Asp, Leu608->Thr, Ala618->Gly, Asp624->Gly, Gln627->Glu, Thr629->Asn, Ser639->Thr and Asp640->Glu.


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Table 2. Serotype, antibiotic susceptibility and amino acid substitutions for three pbp genes in S. pneumoniae showing high MICs of ampicillin and cefotaxime

 
Of 15 strains with a cefotaxime MIC of ≥2 mg/L, 13 strains possessed amino acid substitutions in the pbp2x gene that changed Ser337-The338-Met339-Lys340 (STMK) to Ser-Ala-Phe-Lys (SAFK). In all of the tested strains, no unknown amino acid substitutions were observed in the pbp1a, pbp1b and pbp2a genes.

Figure 1 shows the PFGE patterns of 16 H-PRSP strains listed in Table 2. PFGE was performed according to each serotype of these strains. Serotype 19F (n = 6) was most common, followed by two strains each of 14, 23F, 6A, 6B and non-typeable. Comparisons based on a dendrogram indicated a high similarity among strains of serotypes 19F and 14, but apparent differences in all other pairs of serotype strains.



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Figure 1. PFGE patterns of chromosomal DNA from H-PRSP digested with Apa1 restriction enzyme. These strains show ampicillin MICs ≥4 mg/L, or cefotaxime MICs ≥2 mg/L and possess several additional amino acid substitutions in pbp2x (n = 15) or pbp2b (n = 1) genes compared with the common PRSPs. Lanes: M, {lambda} ladder molecular size marker; 1, ARD-963; 2, ARD-691; 3, ARD-850; 4, ARD-2337; 5, ARD-2098; 6, ARD-1346; 7, ARD-470; 8, ARD-986; 9, ARD-1152; 10, ARD-1268; 11, ARD-213; 12, ARD-1850; 13, ARD-15; 14, ARD-724; 15, ARD-241; 16, ARD-1334.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resistance to various chemotherapeutical agents in S. pneumoniae, a leading cause of respiratory infections, has become a major worldwide problem. In Japan, according to the results based on our PCR methods, the causative gPISP and gPRSP in meningitis reached 37.3 and 41.5%, respectively in 2002.4 Nearly 70% of these isolates also possessed mef(A) or erm(B) genes mediating macrolide resistance. Several reasons for the rapid increase in resistance were considered. One reason was that almost all of the oral cephalosporin antibiotics prescribed to outpatients obtain low serum concentrations and tissue distribution. Basically, circumstances differ from those in Europe and the US, where amoxicillin/clavulanic acid, which obtains high serum concentrations, is recommended as the first choice oral antibiotic for paediatric outpatients with RTIs. It follows that these differences in the prescribing patterns are reflected in the PBP gene alterations in PRSP and PISP. Non-susceptible strains with an abnormal pbp2x gene, resulting in intermediate-resistance to cephalosporin antibiotics, are isolated at a high rate in Japan, while strains with an abnormal pbp2b are more prevalent in the US.9

The relatively high population density in Japan is a secondary factor, which results in easy transmission of resistance among young children.

On a global basis, with the goal of preventing pneumococcal infections in children, large-scale clinical trials of 7-valent conjugate vaccine have been conducted in a number of countries, and vaccination programmes have already become compulsory in some countries.5 However, the S. pneumoniae serotype distributions are known to vary between developing and developed countries, and with patient's age and variety of infection.

In Japan, a clinical trial of a 7-valent conjugate vaccine for children has been in progress since 2004. No recent epidemiological data regarding the serotypes in RTIs, which are fundamental for vaccine introduction, are available except the data of the Nationwide Surveillance for Bacterial Meningitis.

As described in the results, the proportion of gPRSP was as high as 52.3%, while the proportion of gPISP was confirmed as 39.3%, and the majority of them with vaccine serotypes. The 7-valent conjugate vaccine covered 70.9% of all the isolates, but 89.8%, if limited to gPRSP including H-PRSP.

Several data have been reported regarding a change in serotype after vaccination, and the increase in types 3 and 6A is of particular concern.10 A rapid change to a 13-valent conjugate vaccine is required.

Finally, to ensure the effectiveness of the vaccination, it would be ideal to add serotypes 10 and 22, which are common in severe infections in adults, to serotypes 6A, 3 and 23A.


    Acknowledgements
 
We are sincerely grateful to the physicians in the ARD study group, which was organized for this study. This work was partially supported by two grants, one from the Ministry of Health, Labour, and Welfare of Japan (The Research Project for Emerging and Re-emerging Infectious Diseases, April 2003 to March 2005) and the other from the Japanese Ministry of Education, Culture, Sports, Science and Technology (the 21st Century COE program).

The members of the ARD Study Group are: Satoshi Iwata (Head; National Tokyo Medical Center); Hiroko Endo, Reiko Takayanagi, Mika Numata and Shinobu Ishizawa (Tohoku Rosai Hospital); Keisuke Sunakawa and Tomohiro Oishi (Kitasato University School of Medicine); Shigeru Ohnari (Nakafukawa Pediatric Clinic); Naohisa Kawamura (Osaka Rosai Hospital, Osaka Medical College); Haruo Kuroki (Nagatsu-kai Saitoh Hospital); Ritsuko Sakai (Sakai Clinic); Takeshi Tajima and Eiich Nakayama (Hakujikai Memorial Hospital); Masahiko Nitta and Takao Morinobu (Osaka Medical College, Seikeikai Hospital); and Koichi Shimizu (Saiseikai Ibaraki Hospital).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1. Ubukata K. Problems associated with high prevalence of multidrug-resistant bacteria in patients with community-acquired infections. J Infect Chemother 2003; 9: 285–91.[CrossRef][Medline]

2. Ubukata K, Iwata S, Sunakawa K. In vitro activities of new ketolide, telithromycin, and eight other macrolide antibiotics against Streptococcus pneumoniae having mefA and ermB genes that mediate macrolide resistance. J Infect Chemother 2003; 9: 221–6.[CrossRef][Medline]

3. Bergmann C, Chi F, Rachid S et al. Mechanisms for penicillin resistance in Streptococcus pneumoniae: penicillin-binding proteins, gene transfer, and cell wall metabolism. In: Tuomanen EI, Mitchell TJ, Morrison DA et al., eds. The Pneumococcus. Washington, DC: ASM Press, 2004; 339–49.

4. Ubukata K, Chiba N, Hasegawa K et al. Antibiotic susceptibility in relation to penicillin-binding protein genes and serotype distribution of Streptococcus pneumoniae strains responsible for meningitis in Japan, 1999 to 2002. Antimicrob Agents Chemother 2004; 48: 1488–94.[Abstract/Free Full Text]

5. Centers for Disease Control and Prevention. Preventing pneumococcal disease among infants and young children. Recommendations of the Advisory Committee on Immunization Practices. MMWR Morb Mortal Wkly Rep 2000; 49: 1–38.[Medline]

6. Hausdorff WP, Feikin DR, Klugman KP. Epidemiological differences among pneumococcal serotypes. Lancet Infect Dis 2005; 5: 83–93.[ISI][Medline]

7. Yano H, Suetake M, Kuga A et al. Pulse-field gel electrophoresis analysis of nasopharyngeal flora in children attending a day care centre. J Clin Microbiol 1999; 38: 625–9.[ISI]

8. Kosowska K, Jacobs MR, Bajaksouzian S et al. Alterations of penicillin-binding proteins 1A, 2X, and 2B in Streptococcus pneumoniae isolates for which amoxicillin MICs are higher than penicillin MICs. Antimicrob Agents Chemother 2004; 48: 4020–2.[Abstract/Free Full Text]

9. Nagai K, Shibasaki Y, Hasegawa K et al. Evaluations of the primers for PCR to screen Streptococcus pneumoniae isolates, ß-lactam resistance and to detect common macrolide resistance determinants. J Antimicrob Chemother 2001; 48: 915–8.[Abstract/Free Full Text]

10. Whitney CG. Evidence for the emergence of non-vaccine types causing invasive pneumococcal disease in the U.S. In: Abstracts of the Forty-fourth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2004. Abstract 1067, p. 488. American Society for Microbiology, Washington, DC, USA.





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