The optochin susceptibility test is a key method for differentiating Streptococcus pneumoniae from other α-hemolytic streptococci; however, optochin-resistant (Opt r) S. pneumoniae have been reported in the last two decades. In this study, we investigated the isolation frequency of Opt r S. pneumoniae in the North Kyushu area of Japan, and biochemically and genetically characterized Opt r S. pneumoniae clinical isolates. Seven (0.68%) out of 1032 S. pneumoniae isolates collected by the North Fukuoka Infectious Diseases Working Group were found to be Opt r S. pneumoniae. Resistant strains had MICs of optochin 2- to 64- fold higher than susceptible strains, possessed different antimicrobial resistance profiles, and belonged to different serotypes. All the seven Opt r isolates had mutations in the nucleotide sequence code for subunit c of F 0F 1 ATPase. Three isolates had mutations in codon 48 (deduced amino acid substitution of valine with phenylalanine) and two isolates had mutations in codon 49 (substitution of alanine with threonine or serine). Of the remaining two isolates, one had mutation in codon 50 (substitution of phenylalanine with leucine) and the other had mutation in codon 44 (substitution of methionine with isoleucine, which was a novel mutation in this position). From these results, we identified the mutation in the H +-ATPase subunit c gene (atpC) of S. pneumoniae, which was not recognized earlier, and determined that Opt resistance among Japanese pneumococcal isolates is not related to a specific pneumococcal serotype or antimicrobial resistance profile. Furthermore, the results indicate that when α-hemolytic streptococci resistant to optochin are isolated from patients with invasive infectious diseases, such as meningitis and pneumonia, we should perform additional examinations such as bile solubility tests or PCR assays before confirming isolates as viridans streptococci. This is the first report of the characterization of Opt r S. pneumoniae in Japan.
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 [
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 [
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.
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 [
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 (
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. [
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-
turer’s instructions.
The lytA gene was detected by PCR using primers described by Ubukata et al. [
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.
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).
The 16S rDNA (~1.5 kb) was amplified by PCR using the primers 8UA (5’-AGAGTTTGATCMTGGCTCAG- 3’) and 1485B (5’-TACGGTTACCTTGTTACGAC-3’) [
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. [
Transformation in S. pneumoniae R6 was performed as described by Muñoz et al. [
Nucleotide sequence data of partial sequences of atpC genes were deposited in the DDBJ database under the accession numbers AB569578 to AB569584.
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 (
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 (