Antimicrobial susceptibilities and analysis of genes related to penicillin or macrolide resistance in Streptococcus pneumoniae

https://doi.org/10.1016/j.ijantimicag.2004.02.024Get rights and content

Abstract

One hundred and seventy-seven strains of Streptococcus pneumoniae derived from respiratory specimens between 1987 and 2001 were evaluated for their antimicrobial susceptibilities and distribution of genes related to penicillin and macrolide resistance. Resistance rates tended to be higher for the 1996–2001 isolates than for the 1987–1995 isolates for all β-lactams tested. For benzylpenicillin the MIC90 value of the isolates derived between 1996 and 2001 was 1.56 mg/L, while that of strains isolated between 1987 and 1990 was 0.05 mg/L. Furthermore, the number of strains susceptible to macrolides also decreased, but only two strains isolated in 1993 were resistant to levofloxacin of the 177 S. pneumoniae strains tested. When of genes relating to penicillin resistance were analysed using PCR with primers specific to susceptible alleles, although more than 50% of strains from 1987 to 1990 and 1991 to 1995 revealed no mutations in the pbp 1a, 2x and 2b genes, only 30.0% of strains derived between 1996 and 2001 showed no mutations in the pbp gene. Strains having mutations in all three pbp genes (1a, 2x and 2b) by the PCR method increased from only 2.2% in the 1987–1990 derived strains to 27.5% in the 1996–2001 strains. Furthermore, 64.1 and 60.0% of the isolates from 1987 to 1990 and 1991 to 1995, respectively, did not possess either the mefA or ermB by PCR analysis. Conversely, 75.0% of isolates from 1996 to 2001 possessed mefA and/or ermB. These genetic changes may explain the increase in the number of penicillin and macrolide resistant strains. We believe that it is important to evaluate changes in MIC as well as genetic mutations in order to select the most appropriate therapy for S. pneumoniae infections.

Introduction

Streptococcus pneumoniae is one of the major causes of community-acquired pneumonia, acute otitis media, bacterial meningitis and is a frequent cause of bacteraemia. In the past, approximately 80% of patients hospitalised with bacteraemic pneumococcal infections died of their illness [1]. However, with effective antimicrobial agents, mortality has decreased substantially, but remains still high in very young and older age groups [2]. Penicillins and macrolides are still the drug of choice for management of pneumococcal infections in most countries, but since the first description of a strain with diminished susceptibility in 1967, resistance of S. pneumoniae isolates has rapidly spread and resistance to penicillins and macrolides is a world-wide problem [3], [4].

In recent years, several reports have revealed that the number of penicillin- or macrolide-resistant S. pneumoniae strains has increased. A nationwide survey in Japan conducted in 1994–1995 reported that 40.8 and 53.7% of pneumococci were resistant to benzylpenicillin and erythromycin, respectively [5]. Between 1979 and 1987, only 5% of pneumococcal isolates identified in a nationwide surveillance programme were resistant to penicillin in the United States [6]. Subsequent studies have found that more than 30% of S. pneumoniae isolates are not fully susceptible to penicillin in the United States [7], [8]. Furthermore, macrolide-resistant S. pneumoniae strains have increased from 16% in 1994 to 32% in 1999 [9]. Treatment of community-acquired pneumonia with β-lactams or macrolides thus occasionally fails because of the distribution of resistant S. pneumoniae strains.

The mechanism of resistance to macrolides and penicillins has been elucidated in recent years. Recent studies have identified two important gene families that are responsible for macrolide resistance; erm, which mediates ribosomal modification and mef, which encodes a drug efflux pump in clinical isolates [10], [11]. The mef phenotype confers low-level resistance to 14- and 15-membered macrolides but such strains are susceptible to 16-membered macrolides. On the other hand, strains that possess the erm gene show resistance to all of the macrolides, including 16-membered macrolides.

Mutations of the pbp genes, which encode penicillin binding protein (PBP), are responsible for resistance to β-lactams in S. pneumoniae. In particular, the sequences of pbp1a, pbp2b and pbp2x of penicillin-resistant S. pneumoniae (PRSP) and penicillin-intermediate S. pneumoniae (PISP) were more highly diverged than those of penicillin-susceptible S. pneumoniae (PSSP) [12]. Mutations in pbp2b confer low-level resistance to penicillin and pbp2x-related to cefotaxime-resistance, while adding pbp1a mutations seem to result in high resistance.

We believe that it is important to evaluate changes in the distribution of MIC values and gene mutations related to penicillin and macrolide resistance in S. pneumoniae. In this study, we investigated susceptibility to antimicrobial agents and analysed the genes related to penicillin and macrolide resistance using the PCR method in S. pneumoniae derived from respiratory specimens obtained in our hospital between 1987 and 2001.

Section snippets

Bacterial strains

One hundred and seventy-seven strains of S. pneumoniae, isolated at the Clinical Laboratory Center of Oita University Hospital, between 1987 and 2001, from respiratory specimens such as sputum and intratracheal sputum were used in this study.

Antimicrobial agents

Benzylpenicillin (PCG; Meiji Seika Kaisha Ltd., Tokyo, Japan), cefaclor (CCL; Shionogi & Co. Ltd., Osaka Japan), cefotiam (CTM; Takeda Chemical Industries Ltd., Osaka), ceftizoxime (CZX; Fujisawa Pharmaceutical Co. Ltd., Tokyo), cefditoren (CDTR; Meiji

Susceptibility of clinically isolated S. pneumoniae to antimicrobial agents

The susceptibility rates of S. pneumoniae isolated between 1987 and 2001 to eleven antimicrobial agents are shown in Table 1. Resistance rates tended to be higher in the 1996–2001 isolates compared with the 1987–1990 and 1991–1995 isolates for all tested agents, except for LVFX. The isolates derived from 1996 to 2001 showed particularly high resistance rates for PCG, CCL, CTM and EM. Resistance rates were also higher against CZX. CDTR, CFPN and FRPM had good antimicrobial activity but

Discussion

In the present study, we investigated the susceptibility of S. pneumoniae to various antimicrobial agents, including PCG and EM, the distributions of mefA and ermB genes and mutations of the pbp1a, 2x and 2b genes. Additionally, we compared the changes in MIC and distribution of the antimicrobial-resistance genes between 1987 and 2001.

Several reports have indicated a high prevalence of pneumococcal resistance to penicillin as well as other antibiotics, such as cephalosporins and macrolides. A

Acknowledgements

We thank Masumi Ikuta, Mami Kimoto, Shinji Koga, Hiroyuki Tanamachi, Tamio Ueno and Tadao Nakano for experienced technical assistance. This study was supported in part by a grant from Meiji Seika, Kaisha Ltd., Tokyo, Japan.

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