1. Introduction
Streptococcus pneumoniae colonize the nasopharynx in 20% - 40% of children and 5% - 10% of adults at any time, and cause serious infectious diseases, such as pneumonia, septicemia, meningitis, and otitis media [1]. Five phenotypic characteristics are classically used in the clinical laboratory for the presumptive identification of S. pneumoniae: Gram stain morphology, colony morphology, type of hemolysis, optochin susceptibility, and agglutination with anti-pneumococcal polysaccharide capsule antibodies. Accurate identification is important for ensuring the correct diagnosis and treatment of patients because of the increasing frequency of resistance to penicillin and other antibiotic agents.
The optochin susceptibility test is one of the most important methods for differentiating S. pneumoniae from other a-hemolytic streptococci. Optochin (ethylhydrocupreine hydrochloride) is a quinine analog. It was introduced as a therapeutic agent for treatment of lobar pneumonia in early 20th century. However, its use resulted in severe side effects and a study reported that 4.5% of patients treated with optochin experienced loss of vision [2]. Thus, it was stopped being used as a therapeutic agent. Later, in 1915, optochin was found to be useful for differentiating S. pneumoniae from other a-hemolytic streptococci [3]. The optochin susceptibility test was found to be highly satisfactory and less timeconsuming for the identification of S. pneumoniae compared to bile solubility test; hence, it was adopted in clinical laboratories in 1955 [4]. In 1987, Kontiainen and Sivonen [5] first reported the identification of two clinical isolates of optochin-resistant (Optr) S. pneumoniae in blood samples taken from a 74-year-old man with pneumonia and liver cirrhosis and an 8-month-old child with sepsis from otitis media. Since then, the emergence of Optr S. pneumoniae has been reported from the United States [6-8], Israel [9], Portugal [10,11], Brazil [12], and Argentina [13]. In addition, mutations of the gene encoding subunit c of the Fo complex of transmembrane H+-ATPase were reported to be responsible for optochin resistance [14]. There have been no reports of Optr S. pneumoniae in Japan.
The purpose of this study is to investigate the isolation frequency of Optr S. pneumoniae in Japan, report the biochemical and genetic characteristics of the isolates, and alert clinical microbiologists of the presence of these strains in the community.
2. Materials and Methods
2.1. Bacterial Strains
A total of 1032 presumptive S. pneumoniae isolates were collected by the North Fukuoka Infectious Diseases Working Group (NFIDWG, Fukuoka, Japan). These clinical isolates were recovered mainly from the nasopharynx and sputum samples obtained from 138 medical clinics and hospitals participating in NFIDWG. Each isolate was confirmed to be S. pneumoniae based on its Gram stain morphology, colony morphology, type of hemolysis, optochin susceptibility, and agglutination with anti-pneumococcal polysaccharide capsule antibodies. In cases where optochin resistance was suspected, we conducted further examinations such as the bile solubility test, determination of the presence of the major autolysin gene (lytA) by PCR [15], determining bacterial profiles by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and sequencing of the 16S rRNA genes (1480 to 1485 bases). S. pneumoniae R6 ATCC BAA-255 (uncapsulated derivate of D39) was used as the reference strain.
2.2. Optochin Susceptibility
Optochin disks (5 μg each; Eiken Co., Ltd., Tokyo, Japan) were placed on 5% sheep blood agar plates (Eiken) streaked with the test isolate. The diameter of inhibition zones around the disk was measured after 18 - 24 h incubation at 35˚C - 37˚C in a 5% CO2 atmosphere. Bacterial isolates with a diameter measuring ≥13 mm were tentatively identified as optochin-sensitive, while isolates with a diameter < 13 mm were identified as optochinresistant. The MICs of optochin (Sigma Co. LLC, St. Louis, MO, USA) for 1031 isolates were determined using the plate dilution method with Mueller-Hinton agar (Difco Laboratories, Detroit, MI, USA) containing 5% defibrinated sheep blood (Kohjin Bio Co., Ltd., Saitama, Japan) and an inoculum size of 104 CFU of bacteria. Cell growth was evaluated after incubation for 24 h at 37˚C in a 5% CO2 atmosphere. Bacterial isolates were considered optochin resistant when MICs were ≥4 μg/ml, based on the result of our present study (Figure 1) and a previous report by Pikis et al. [8].
2.3. Bile Solubility, Latex Agglutination, and Capsular Serotyping
Bile solubility and latex agglutination tests were performed using the Slidex Pneumo-Kit (Nippon bioMérieux Co., Ltd., Tokyo, Japan) based on the methods described by Whatmore et al. [16]. Capsular serotyping (the Neufeld Quellung test) was performed using each type or group serum (Statens Serum Institut, Copenhagen, Denmark) at the National Institute of Infectious Diseases, Tokyo, Japan.
2.4. Biotyping
Biotyping was performed using API 20 Strep V7.0 (Nippon bioMérieux) and an automated VITEK 2 Gram Positive Identification (GPI) Card (Nippon bioMérieux) at the Central and Clinical Laboratories in Saga University Hospital by medical technologists who are specialists in clinical microbiology. Cultures were grown anaerobically on 5% sheep blood agar (Eiken) at 35˚C - 37˚C for 22 - 26 h and suspensions were prepared for API 20 Strep V7.0 according to the manufacturer’s instructions. The result was determined by matching data with an API 20 Strep profile list. For preparing the GPI Card, colonies were picked and suspended in a 3.0-ml-sterile salt solution (pH 4.5 - 7.0), which was equivalent to a McFarland’s 0.50 - 0.63 standard according to the manufac-
Figure 1. Distribution of MICs of Optochin in Streptococcus pneumoniae clinical isolates (1032 strains). The MICs are determined by the plate dilution method.
turer’s instructions.
2.5. PCR for the Major Autolysin Gene (lytA)
The lytA gene was detected by PCR using primers described by Ubukata et al. [15]. Using a Biometra T Gradient Thermocycler (Biometra GmbH, Goettingen, Germany), thirty cycles of DNA amplification were performed as follows: denaturation at 94˚C for 15 s, annealing at 50˚C for 30 s, and extension at 72˚C for 30 s. The presence of an amplified 273-bp sequence of the autolysin gene indicated the presence of the lytA gene. The DNA size marker was 100 Base-Pair Ladder DNA (Pharmacia Biotech Co., Ltd., Tokyo, Japan).
2.6. Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) Identification
Identification of isolates by MALDI-TOF MS was performed on a Microflex LT instrument (Bruker Daltonics GmbH, Leipzig, Germany) with FlexControl (version 3.0) software (Bruker Daltonics) for the automatic acquisition of mass spectra in the linear positive mode within a range of 2 - 20 kDa. Colonies were examined by both direct deposition on MSP 96 target plates (Bruker Daltonics) and after a formic acid-acetonitrile extraction step according to the manufacturer’s instructions. According to the criteria proposed by the manufacturer, a result was considered valid (accurate identification to the species level) when the score was >2.0.
2.7. Antibiotic Susceptibility
The MICs to 12 antimicrobial agents were determined using the microdilution broth method following the Clinical and Laboratory Standards Institute (CLSI) guidelines. The antibiotics used in this test were as follows: benzylpenicillin (PCG), ampicillin (ABPC), cefazolin (CEZ), cefotiam (CTM), cefotaxime (CTX), cefpodoxime (CPDX), cefditoren (CDTR), panipenem (PAPM), minocycline (MINO), erythromycin (EM), clindamycin (CLDM), and levofloxacin (LVFX).
2.8. Sequencing of 16S rRNA Gene
The 16S rDNA (~1.5 kb) was amplified by PCR using the primers 8UA (5’-AGAGTTTGATCMTGGCTCAG- 3’) and 1485B (5’-TACGGTTACCTTGTTACGAC-3’) [17]. The purified PCR product was sequenced directly on both strands using a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and a BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems). The primers 519A (5’-CAGCMGCCGCGGTAA- 3’), 519B (5’-ATTACCGCGGCRGCTG-3’), 774A (5’- GTAGTCCACGCTGTAAACGATG-3’), 774B (5’-CATCGTTTACAGCGTGGACTAC-3’), and 907B (5’-CCGTCAATTCMTTTRAGTTT-3’) were used as internal primers for sequencing. Homology search with the 16S rRNA gene sequences were performed against sequences registered in GenBank/EMBL/DDBJ using a basic local alignment search tool (BLAST).
2.9. Cloning and DNA Sequence Analysis of H+-ATPase Subunit c Gene
The H+-ATPase subunit c gene (atpC) of S. pneumoniae was amplified by PCR using primers (sense primer: 136 5’-TAGCGGTTAAAAGTTGACAA-3’; antisense primer: 437 5’-CCCTTTTCTTCTCGTTCC-3’) described by Cogné et al. [18]. After initial denaturation at 95˚C for 2 min, 25 cycles of DNA amplification were performed as follows: denaturation at 95˚C for 1 min, annealing at 54˚C for 2 min, extension at 72˚C for 2 min 30 s, and a final extension at 72˚C for 7 min 30 s. The expected 302-bp fragment was purified using PCR purification columns (GenElute Minus EtBr Spin Columns; Sigma Chemical Co., St Louis, MO, USA) and cloned using a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Transformation was performed using competent E. coli TOP10 cells provided by the manufacturer. A total of three to five white colonies were randomly selected from each clone library for sequence analysis. To prepare a template for sequence analysis, a partial fragment of the cloning vector (PCR II) containing an inserted PCR product was amplified using M13Forward (5’-GTAAAACGACGGCCAG-3’), M13Reverse (5’-CAGGAAACAGCTATGAC-3’), and AmpliTaq Gold DNA polymerase. Primers and dNTP were eliminated from the PCR mixture using an ExoSAP-IT Kit (USB, Cleveland, OH, USA) according to the manufacturer’s instructions and a 1 ml aliquot was used as a template for the sequencing reaction. Sequencing reactions were performed using the M13 primers and a BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems). The nucleic acid sequences were determined using a 3130xl Genetic Analyzer (Applied Biosystems).
2.10. Genetic Transformation
Transformation in S. pneumoniae R6 was performed as described by Muñoz et al. [19]. S. pneumoniae R6 was grown in C medium plus yeast extract (C + Y) [20] to the late exponential phase and frozen at −80˚C after the addition of glycerol to 15%. Frozen stock (200 µl) was added to 4 ml of C + Y medium and incubated for about 2 h to reach competence for transformation. The competent S. pneumoniae R6 cells were diluted 10-fold in C + Y medium and a cloned PCR fragment (atpC gene) of S. pneumoniae SPJ661 was added at final concentrations of 0.1 mg/ml to 500 ml of the diluted cultures. These were then incubated at 30˚C for 1 h and at 37˚C for at least 2 h to allow expression of optochin resistance. Samples were then plated in 100 ml volumes on Mueller-Hinton agar (Difco Laboratories) containing 5% defibrinated sheep blood (Kohjin Bio) and 2 mg/ml optochin (Sigma).
2.11. Nucleotide Sequence Accession Numbers
Nucleotide sequence data of partial sequences of atpC genes were deposited in the DDBJ database under the accession numbers AB569578 to AB569584.
3. Results
3.1. Isolation Frequency and Biochemical Characteristics of Optochin-Resistant S. pneumoniae
A total of 1032 strains conclusive or presumptive as S. pneumoniae were collected by NFIDWG from patients with invasive diseases, such as pneumonia, septicemia, meningitis, and otitis media. Of these, 1025 isolates (99.32%) exhibited an optochin MIC of ≤2 mg/ml and seven isolates (0.68%) exhibited an MIC of ≥4 mg/ml (three isolates, 4 mg/ml; 1 isolate, 32 mg/ml; three isolates, ≥128 mg/ml) using the plate dilution method (Figure 1, Table 1). Table 1 shows that three of the seven optochin-resistant strains were isolated from the nasal discharges of children, while four strains were isolated from the sputum of elderly individual persons. As shown in Table 2, all isolates presented typical Gram staining characteristics, a-hemolysis on blood agar plates, bile solubility, and agglutination reactions with sera targeting pneumococcal capsular polysaccharides. In addition, all isolates contained the lytA gene and exhibited autolysis.
However, they were not confirmed as S. pneumoniae using API Strep 20 V.7.0 because they had biochemical alterations with regards to optochin resistance, a loss of inulin and starch utilization, and a loss of arginine hydrolysis and pyrrolidone arylamidase (Table 3). Table 2 shows that they were identified as S. pneumoniae using the extraction method based on MALDI-TOF MS analysis. Mass spectra were not obtained for strain SPJ1298 using the direct method because it produced mucoid colonies. Mass spectra were also not obtained for another three isolates probably because of autolysis, when the colonies were analyzed on day 1 after being cultured on blood agar plates (Table 2). Isolates belonged to different serotypes (Table 2) and exhibited different antimicrobial resistance profiles (Table 1). Although all Optr isolates were susceptible to PCG and LVFX, six of the seven isolates were resistant to EM (Table 1). In addition, five isolates were CPDX intermediates or CPDX resistant, and four isolates were resistant to CLDM (Table 1). Finally, all isolates were confirmed to be S. pneumoniae by the 16S rRNA gene analysis (Table 2).
3.2. Genetic Characteristics of Optochin-Resistant S. pneumoniae
Table 4 shows that all the seven Optr isolates had mutations in the nucleotide sequence coding for subunit c of F0F1 ATPase. Three isolates (SPJ48, 492, and 1298) had mutations in codon 48 (GTT to TTT, deduced amino acid substitution of valine with phenylalanine) and two isolates (SPJ743 and 1331) had mutations in codon 49 (GCC to ACC or TCC, substitution of alanine with threonine or serine). Of the remaining two isolates, one (SPJ246) had mutation in codon 50 (TTT to CTT, substitution of phenylalanine with leucine) and the other