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INTRODUCTION
Methicillin-susceptible Staphylococcus aureus (MSSA), Streptococcus pyogenes, Streptococcus pneumoniae and Haemophilus influenzae are major causative pathogens of respiratory infectious disease and established causes of pneumonia, otitis media and paranasal sinusitis. In Japan and other Asian countries, S. pneumoniae has been rapidly developing β-lactam resistance by amino-acid substitutions in penicillin-binding proteins (PBPs). Isolates of these new strains also frequently show resistance to macrolides conferred by macrolide-resistant genes (1-3). Macrolide resistance in S. pneumoniae is conferred by 23S ribosome methylation by ErmB and/or drug efflux systems by MefA (4). Similarly, encounters with ermA-, ermB-, ermC- or mefA-positive MSSA and S. pyogenes have been growing more common in clinical settings. Clinicians in Japan have also been isolating new strains of H. influenzae, β-lactamase-negative ampicillin resistant strains (BLNAR), with increasing frequency (1, 5). Taking all of these developments together, it seems certain that respiratory pathogens are acquiring β-lactam and macrolide resistance. This makes it all the more important to examine the antibacterial activity against multidrug-resistant bacteria isolated from patients with respiratory tract infections (2, 5-9).
In this study we examine the in vitro activities of 7 antibiotics, oral cephems (cefditoren, cefcapene and cefdinir), macrolide (erythromycin), ketolide (telithromycin) and controls (benzylpenicillin and ampicillin) against clinical isolates of four major respiratory pathogens (MSSA, S. pyogenes, S. pneumoniae, and H. influenzae) collected in Japan between 2002 and 2003.
MATERIALS AND METHODS
Clinical isolates
MSSA, S. pyogenes, S. pneumoniae and H. influenzae, which were isolated from the respiratory tract and collected at 66 institutions (20 university hospitals; 34 local central hospitals; 4 clinics; 8 general practitioners) in all over the Japan between 2002 and 2003, were used in this study. Forty-three isolates of MSSA and 49 isolates of S. pyogenes strains were randomly selected. S. pneumoniae was classified in three categories, based on the mutations of the pbp genes associated with β-lactam resistance. As a result, the following isolates were studied: 34 isolates of genotypic penicillin-susceptible S. pneumoniae (gPSSP) with no mutations in the pbp1a, 2x or 2b genes; 47 isolates of genotypic penicillin-intermediate-resistant S. pneumoniae (gPISP) with mutations in two of three pbp genes (1a and 2x, 1a and 2b or 2x and 2b) and 68 isolates of genotypic penicillin-resistant S. pneumoniae (gPRSP) with mutations in all three pbp genes, as described by Ubukata et al. (9). The H. influenzae isolates were classified into two variants. The first, BLNAR, had M377I, S385T, L389F and N526K mutations of the ftsI gene encoding PBP3 without carrying the TEM-1 gene encoding β-lactamase. The second, β-lactamase-negative ampicillin-susceptible H. influenzae (BLNAS) had no mutations of the ftsI gene and did not carry the TEM-1 gene. Twenty-five BLNAS isolates and 24 BLNAR isolates were selected for inclusion in this study.
Detection of drug-resistance genes by PCR
Mutations in the pbp1a, 2x and 2b genes and the presence of the mefA and ermB genes in S. pneumoniae were detected by a penicillin-resistant S. pneumoniae detection kit (Wakunaga Pharmaceutical, Osaka, Japan). Mutations in the ftsI gene and the presence of the TEM-1 gene in H. influenzae were detected using an H. influenzae gene detection kit (Wakunaga Pharmaceutical). The presence of the ermA and ermC genes in MSSA, and the presence of the mefA and ermB genes in S. pyogenes, were detected by conventional PCR methods (6, 10).
Antibiotic susceptibility test
Cefditoren and ampicillin were supplied by Meiji Seika Kaisha, Ltd., Tokyo, Japan. Cefdinir was purchased from Kemprotec Ltd., Middlesborough, U.K. Erythromycin and benzylpenicillin was purchased from Sigma-Aldrich Japan. Cefcapene was synthesized in our laboratory. Telithromycin was purified from the commercial product (Aventis Pharma Japan, Tokyo, Japan). Minimum inhibitory concentrations (MICs) were determined by the agar dilution method with 2-fold serial dilutions of antibiotics. The following Mueller-Hinton agar (MHA, Becton, Dickinson and Company, Sparks, MD, U.S.A.) preparations were used for the drug susceptibility tests: MHA alone for MSSA; MHA supplemented with 5% defibrinated equine blood for S. pneumoniae; MHA supplemented with 5% defibrinated equine blood heated until chocolate for H. influenzae, and MHA supplemented with 5% defibrinated ovine blood for S. pyogenes. Each isolate was inoculated at 104 CFU/spot on each agar plate. The MIC was determined as the minimum drug concentration showing no bacterial growth after 18-20 hr of incubation at 35℃. S. aureus ATCC29213, S. pneumoniae ATCC49619, H. influenzae ATCC49247 and ATCC49766 were used to control the accuracy of the MIC determinations. S. pneumoniae ATCC49619 was used as a control in the measurement of the susceptibility of S. pyogenes isolates. As for the breakpoint values of antibiotics for pneumoniae, we referred to the section of pneumoniae in the journal issued by the Sensitivity Determination Committee for Antibiotics, Japanese Society of Chemotherapy, except for benzylpenicillin that was not provided this journal. Judging from the breakpoint value, we calculated the resistance or susceptibility rate of each antibitotic against pathogens.
RESULTS
Antibacterial activity against MSSA
Of the 43 MSSA isolates used in this study, 10 isolates (23.3%) and 3 isolates (7.0%) carried either ermA or ermC, respectively, while none of the strains carried both genes. Telithromycin showed the most potent antibacterial activity against MSSA, followed by cefdinir, cefditoren, and cefcapene in descending order (Table 1). The antibacterial activity of erythromycin against MSSA was markedly attenuated by the presence of the erm genes, whereas the MIC90 of telithromycin was only 0.06 mg/L, even in the presence of the erm genes, with no cross-resistance to erythromycin.
Antibacterial activity against S. pyogenes
As shown in Table 2, 6 isolates (12.2%) and 17 isolates (34.7%) of the S. pyogenes strains carried the ermB and mefA genes, respectively. The MIC of the cephem antibiotics was no more than 0.015 mg/L against all the isolates of S. pyogenes tested. The MIC90 of telithromycin was increased when the isolates carried either ermB or mefA. Especially high resistance against telithromycin (MIC=64 mg/L) was observed in 4 of 6 isolates carrying the ermB gene.
Antibacterial activity against S. pneumoniae
Subdivision of the gPSSP, gPISP, and gPRSP strains based on the presence of the ermB and mefA genes revealed that S. pneumoniae carried either ermB or mefA at a high frequency (Table 3). The β-lactams showed potent antibacterial activity against gPSSP, and cefditoren showed the strongest antibacterial activity against gPSSP, gPISP, and gPRSP of all 3 β-lactam antibiotics. The presence of the ermB and mefA genes within S. pneumoniae strains did not affect the activity the β-lactams. Telithromycin and erythromycin showed no attenuation in antibacterial activity even in the presence of a mutated pbp gene, while telithromycin exerted the strongest antibacterial activities against all three of the S. pneumoniae strains. However, the antibacterial activity of telithromycin was weakened in the presence of either the ermB or mefA gene.
Antibacterial activity against H. influenzae
Cefditoren and cefcapene showed stronger antibacterial activity than cefdinir, telithromycin, and erythromycin against BLNAS (Table 4). The antibacterial activities of the oral cephems were weaker against BLNAR than against BLNAS, whereas the MIC90 of cefditoren was 0.5 mg/L, the most potent level measured among all the antibiotics tested in this study. Telithromycin showed an equal MIC90 against BLNAR and BLNAS, and the same result was obtained for erythromycin.
DISCUSSION
The incidence of drug-resistant pathogens differs greatly between countries, presumably in accordance with differences in the dosage and usage of antibiotics. In this study we focused on the antibacterial activities of three different types of antibiotics (cephem, macrolide and ketolide) against recently isolated pathogens from respiratory tract infections carrying various drug-resistance genes in the Japanese population. The different frequencies of drug-resistant pathogens between Japan and neighboring Asian countries have also been considered.
In S. aureus, ribosome methylation and the presence of drug efflux systems have been shown to confer resistance against macrolide antibiotics (4). The isolates that exhibited low susceptibility to erythromycin without the erm gene in this study may have overexpressed multidrug efflux systems such as MsrA (11). Interestingly, all MSSA strains examined in this study were susceptible to telithromycin, even when they were positive for the presence of the erm gene. The expression of the erm gene can be inducible or constitutive, and MSSA is resistant to telithromycin when the expression is constitutive (12). Thus, we know that the isolates used in this study might harbor the inducible type of gene expression. The rate of MSSA resistance to telithromycin in Asia, 20%, is higher than the rates in Europe and the U.S.A. (13).
β-lactam resistance has not yet been reported in S. pyogenes, but macrolide-resistant strains of this pathogen have been frequently isolated (6-8, 13, 14). Some of the S. pyogenes isolates carrying ermB have become highly resistant to telithromycin (MIC >32 mg/L) (6-8). In our study, telithromycin and erythromycin showed reduced antibacterial activity against S. pyogenes isolates carrying either ermB or mefA. The incidence of telithromycin-resistant S. pyogenes (MIC ≥4 mg/L) differs from region to region, presumably due to the different usage of antibiotics. For example, the incidence in Japan stands at less than 1%, while the incidences in Korea and Europe are generally above 10% (13).
As previously reported, we categorized S. pneumoniae into gPRSP or gPISP based on mutations in the pbp gene (9). ermB and mefA have been found to be present at high frequencies in recent Japanese isolates of PISP and PRSP (2, 3). Our results also revealed many gPRSP and gPISP isolates carrying either ermB or mefA. In Korea and Hong Kong, PRSP accounts for ≥60% of clinical isolates of S. pneumoniae, and 50% or more of these isolates carry either ermB or mefA (3). It thus comes as no surprise that the MIC of telithromycin has already risen to 4 mg/L against newly emerging strains of S. pneumoniae in Taiwan (15) and to 2-8 mg/L against strains in other countries (7).
Just as Japan is witnessing a rising prevalence of BLNAR and falling prevalence of β-lactamase-producing strains, the very opposite trends are being observed in Korea (1, 3). It might be reasonable to assume that penicillin is more frequently prescribed in Korea than in Japan. Accordingly, the oral cephems such as cefditoren, an agent stable against β-lactamase in H. influenzae, may be useful and effective in both of these countries. Although macrolide-resistant genes such as erm or mef have not yet been found in H. influenzae, the antibacterial activity of telithromycin and erythromycin was lower than that of cefditoren against the H. influenzae isolates analyzed in the present study. Peric et al. demonstrated that more than 98% of H. influenzae strains have a macrolide efflux mechanism (16). Thus, the H. influenzae isolates investigated in this study might also express efflux pumps, such as AcrAB (17).
In conclusion, the variable antibacterial activity against the clinical pathogens in the presence of drug-resistant genes demonstrated in this study suggests that the inappropriate prescription of antibiotic agents may lead to clinical failure. It will be important to choose suitable antibiotics and to make the most of the ability of the prescribed agents in order to prevent the further emergence of drug-resistant pathogens.
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