Optochin Resistance in Streptococcus Pneumoniae: Mechanism, Significance, and Clinical Implications
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582 Optochin Resistance in Streptococcus pneumoniae: Mechanism, Significance, and Clinical Implications Andreas Pikis,1,2 Joseph M. Campos,3,4,5,6 1Vaccine and Therapeutic Development Section, Oral Infection William J. Rodriguez,2,4,a and Jerry M. Keith1 and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland; Departments of 2Infectious Diseases and 3Laboratory Medicine, Children’s National Medical Center, and Departments of 4Pediatrics, 5Pathology, and 6Microbiology/Tropical Medicine, George Washington University Medical Center, Washington, DC Downloaded from https://academic.oup.com/jid/article/184/5/582/808236 by guest on 23 September 2021 Traditionally, Streptococcus pneumoniae is identified in the laboratory by demonstrating susceptibility to optochin. Between 1992 and 1998, 4 pneumococcal isolates exhibiting op- tochin resistance were recovered from patients at Children’s National Medical Center. Three of the 4 isolates consisted of mixed populations of optochin-resistant and -susceptible organ- isms. Both subpopulations had identical antibiograms, serotypes, and restriction fragment profiles. The other isolate was uniformly resistant to optochin. Resistant strains had MICs of optochin 4–30-fold higher than susceptible strains, belonged to different serotypes, and had dissimilar restriction fragment profiles, indicating clonal unrelatedness. Resistance arose from single point mutations in either the a-subunit (W206S) or the c-subunit (G20S, M23I, and A49T) of H+-ATPase. There is speculation of a possible association between exposure to antimalarial drugs and evolution of optochin resistance. a-Hemolytic streptococci resistant to optochin, particularly invasive isolates, should be tested for bile solubility or with an S. pneumoniae DNA probe before identification as viridans streptococci. Streptococcus pneumoniae remains a major cause of human twentieth century as a chemotherapeutic agent for the treatment morbidity and mortality, particularly at both extremes of the of lobar pneumonia. However, serious side effects coupled with age spectrum [1]. The recent emergence of pneumococci resis- treatment failures quickly terminated the therapeutic use of this tant to penicillin, third-generation cephalosporins, and other agent [5]. Although optochin susceptibility was first described antimicrobial agents has raised concerns about treatment of for differentiating pneumococci from other a-hemolytic strep- patients with serious pneumococcal infections [2]. Because of tococci in 1915 [6], the test was virtually unused by laboratories the increasing frequency of antimicrobial resistance, accurate until the mid-1950s [7–9]. During the first 30 years of its use, identification and antimicrobial susceptibility testing are crucial there were no reports of optochin-resistant pneumococci. Since for correct diagnosis and treatment of patients. then, there have been sporadic reports of optochin-resistant Differentiation of S. pneumoniae from other viridans strep- pneumococci [10–12]. Investigators from Spain have cloned, tococci depends on demonstrating optochin susceptibility, bile sequenced, and characterized Hϩ-ATPase c-subunit as the gene solubility, reaction with a specific DNA probe, or detection of encoding the optochin determinant [13]. species-specific capsular polysaccharides [3]. Most clinical mi- In the United States, only 2 optochin-resistant strains have crobiology laboratories today depend on the optochin suscep- been reported. We reported the first isolate in 1997 from Wash- tibility test [4]. ington, DC [14], and Borek et al. [15] reported another from Optochin, a quinine analogue, was introduced early in the Chicago later that year. We since have recovered 3 additional optochin-resistant strains. The purpose of this communication Received 12 March 2001; revised 21 May 2001; electronically published is to report our characterization of the optochin resistance phe- 26 July 2001. notype of these strains and to alert physicians and clinical mi- Presented in part: American Pediatric Society and Society for Pediatric crobiologists to the existence of these strains in the community. Research meeting, Washington, DC, May 1997 (abstract 755; Pediatr Res 1997; 41:128A); Pediatric Academic Societies and American Academy of Pediatrics joint meeting, Boston, May 2000 (abstract 1613; Pediatr Res 2000; 47:273A). Materials and Methods a Present affiliation: Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, Maryland. Patients and strains. From 1 June 1992 through 31 May 1998, Reprints or correspondence: Dr. Andreas Pikis, National Institute of Den- 587 pneumococcal isolates were recovered from ordinarily sterile tal and Craniofacial Research, National Institutes of Health, Bldg. 30, Rm. 528, 30 Convent Dr., MSC 4350, Bethesda, MD 20892-4350 (apikis@dir body sites of patients treated at Children’s National Medical Center .nidcr.nih.gov). (Washington, DC). Three (0.5%) isolates were either uniformly re- sistant to optochin (strain 310) or consisted of mixed populations The Journal of Infectious Diseases 2001;184:582–90 ᭧ 2001 by the Infectious Diseases Society of America. All rights reserved. of optochin-susceptible and -resistant colonies (strains 606a and 0022-1899/2001/18405-0009$02.00 654; figure 1). An additional isolate exhibiting optochin-resistant JID 2001;184 (1 September) Optochin Resistance in S. pneumoniae 583 Downloaded from https://academic.oup.com/jid/article/184/5/582/808236 by guest on 23 September 2021 Figure 1. Optochin test (disk diffusion method) of Streptococcus pneumoniae isolates 310 and 654. Isolate 310 had no inhibitory zone around the optochin disk. Isolate 654 had a 114-mm inhibitory zone with optochin-resistant colonies within the zone. Isolates 606a and HV109 had results similar to those for isolate 654. variants (strain HV109) was recovered from the nasopharynx of a from overnight 5% sheep blood agar cultures. The suspensions were patient during a study that investigated the prevalence of penicillin- divided into 2 tubes. Deoxycholate reagent (2%; 0.5 mL; Becton resistant S. pneumoniae colonization in human immunodeficiency Dickinson Microbiology Systems) was added to one tube, and nor- virus–infected children. Isolates with optochin-resistant variants mal saline (0.5 mL) was added to the other. Tubes were incubated formed distinct colonies within the 14-mm diameter inhibitory zone at room temperature for 15 min. Complete clearing of the suspension that differentiates optochin susceptibility from resistance. Subcul- in the deoxycholate tube was indicative of S. pneumoniae. tures of these colonies exhibited uniform resistance to optochin. Serotyping. Serotyping was done with the Quellung (Neufeld) We used 2 optochin-susceptible clinical isolates and an unencap- reaction (capsular swelling) test. sulated reference strain (R6) as controls. Table 1 shows the char- Genomic DNA analysis. Preparation of agarose plugs containing acteristics of the study isolates. genomic DNA and analysis by pulsed-field gel electrophoresis Antibiotic susceptibility testing. MICs of optochin and several (PFGE) after digestion with SmaI were done as described elsewhere antimicrobial agents were determined by the agar dilution method. [16]. Bacterial suspensions were prepared in 0.9% NaCl from overnight Polymerase chain reaction (PCR). Based on the sequence re- cultures on 5% sheep blood agar and were adjusted to match the ported by Fenoll et al. [13], we designed primers 663 (5-TCGA- 0.5 McFarland turbidity standard. Suspensions were inoculated AAAGTGGATCAACAACTATCC-3) and 1016 (5-TGGGAAA- onto Mueller-Hinton agar containing 3% lysed horse blood and GAAGAAGTAACAAACTCG-3) to amplify the DNA fragment varying concentrations of optochin or selected antimicrobial encoding the ATPase c-subunit from the pneumococcal strains ex- Њ agents. Cultures were incubated at 35 C for 20–24 h in a 5% CO2 amined in this study. The components of the amplification mixtures atmosphere. The MIC was defined as the lowest concentration of (100 mL) were as follows: 5 U of Pfu DNA polymerase (Stratagene), optochin or antimicrobial agent that inhibited visible growth of 1ϫ reaction buffer provided by the manufacturer, 20 mM each of the isolate. the 4 dNTPs, 100 ng of DNA, and 250 ng of each primer. Ampli- Optochin susceptibility test by disk diffusion method. Optochin fication was done in a thermal cycler (model 9600; Perkin-Elmer disks (6 mm; Becton Dickinson Microbiology Systems) were ap- Instruments). After an initial 2-min incubation at 95ЊC, the mixture plied to trypticase soy agar plates with 5% sheep blood streaked was subjected to 30 cycles of amplification under the following con- with the organism being tested. After overnight incubation at 35ЊC ditions: 95ЊC, 1 min; 58ЊC, 1 min; 72ЊC, 2 min; and a final 10-min Њ ina5%CO2 atmosphere, inhibitory zones around the disk were runoff at 72 C. After purification, the PCR product was ligated into measured. Isolates displaying zones у14 mm in diameter were iden- pCR-Blunt vector (Invitrogen), and the ligation products were trans- tified as S. pneumoniae. Isolates displaying zones !14 mm were formed into Escherichia coli TOP10 competent cells. E. coli trans- tested for bile solubility. formants were selected on LB agar containing 50 mg/mL kanamycin. Bile solubility. Heavy bacterial suspensions (1 mL) matching the DNA sequence analysis. Sequencing was done with the di- 2.0 McFarland turbidity standard were prepared in normal saline